Patent Application
20080187969
The present invention relates to oligonucleotides useful for priming the amplification of nucleic acid molecules.
Gene expression analysis often involves amplification of starting nucleic acid molecules. Amplification of nucleic acid molecules may be accomplished by reverse transcription (RT), in vitro transcription (IVT) or the polymerase chain reaction (PCR), either individually or in combination. The starting nucleic acid molecules may be mRNA molecules, which are amplified by first synthesizing complementary cDNA molecules, then synthesizing second cDNA molecules that are complementary, to the first cDNA molecules, thereby producing double stranded cDNA molecules. The synthesis of first strand cDNA is typically accomplished using a reverse transcriptase and the synthesis of second strand cDNA is typically accomplished using a DNA polymerase. The double stranded cDNA molecules may be used to make complementary RNA molecules using an RNA polymerase, resulting in amplification of the original starting mRNA molecules. The RNA polymerase requires a promoter sequence to direct initiation of RNA synthesis. Complementary RNA molecules may, for example, be used as a template to make additional complementary DNA molecules.
Amplification of nucleic acid molecules requires the use of oligonucleotide primers that specifically hybridize to one or more target nucleic acid molecules in the starting material. Each oligonucleotide primer may include a promoter sequence that is located 5′ to the hybridizing portion of the oligonucleotide that hybridizes to the target nucleic acid molecule(s). If the hybridizing portion of an oligonucleotide is too short, then the oligonucleotide does not stably hybridize to a target nucleic acid molecule and priming and subsequent amplification does not occur. Also, if the hybridizing portion of an oligonucleotide is too short, then the oligonucleotide does not specifically hybridize to one or a small number of target nucleic acid molecules, but non-specifically hybridizes to numerous target nucleic acid molecules.
Amplification of a complex mixture of different target nucleic acid molecules (e.g., mRNA molecules) typically requires the use of a population of numerous oligonucleotides having different nucleic acid sequences. The cost of the oligonucleotides increases with the length of the oligonucleotides. In order to control costs, it is preferable to make oligonucleotide primers that are no longer than the minimum length required to ensure specific hybridization of an oligonucleotide to a target sequence.
It is often undesirable to amplify highly expressed RNAs (e.g., ribosomal RNAs). For example, in gene expression experiments that analyze expression of genes in blood cells, amplification of numerous copies of abundant globin mRNAs, or ribosomal RNAs, may obscure subtle changes in the levels of rare mRNAs. Thus, there is a need for populations of oligonucleotide primers that selectively amplify desired nucleic acid molecules within a population of nucleic acid molecules (e.g., oligonucleotide primers that selectively amplify all mRNAs that are expressed in a cell except for the most highly expressed mRNAs). In order to reduce the cost of synthesizing the population of oligonucleotides, the hybridizing portion of each oligonucleotide should be no longer than necessary to ensure specific hybridization to a desired target sequence under defined conditions.
In one aspect, the present invention provides methods for selectively amplifying a target population of nucleic acid molecules (e.g., all mRNA molecules expressed in a cell type except for the most highly expressed mRNA species). The methods of this aspect of the invention each include the step of using a population of oligonucleotides to prime the amplification of a target population of nucleic acid molecules within a larger population of nucleic acid molecules. The population of oligonucleotides is selected based on its ability to hybridize under defined conditions to a first subpopulation of a target nucleic acid population, but not to hybridize under the defined conditions to a second subpopulation of the target nucleic acid population.
In another aspect, the present invention provides populations of oligonucleotides including the nucleic acid sequences set forth in SEQ ID NOS:1-933. These oligonucleotides can be used, for example, to prime the synthesis of cDNA molecules complementary to mRNA molecules isolated from mammalian blood without priming the synthesis of cDNA molecules complementary to globin mRNA or ribosomal RNA molecules. In some embodiments, each oligonucleotide in the population of oligonucleotides further comprises a defined sequence portion located 5′ to the hybridizing portion. In one embodiment, the defined sequence portion comprises a transcriptional promoter, which may be used as a primer binding site in PCR amplification, or for in vitro transcription. In another embodiment, the defined sequence portion comprises a primer binding site that is not a transcriptional promoter. For example, in some embodiments the present invention provides populations of oligonucleotides wherein a transcriptional promoter, such as the T7 promoter (SEQ ID NO:934), is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. In further embodiments, the present invention provides populations of oligonucleotides wherein the defined sequence portion comprises at least one primer binding site which is useful for priming a PCR synthesis reaction, and which does not include an RNA polymerase promoter sequence. A representative example of a defined sequence portion for use in such embodiments is provided as 5′ CCGAACTACCCACTTGCATT 3′ (SEQ ID NO:956), which is preferably located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933.
In another aspect, the present invention provides a reagent for selectively amplifying a target population of nucleic acid molecules (e.g., all mRNA molecules expressed in a cell type except for the most highly expressed mRNA species). The reagent of this aspect of the invention comprises a population of oligonucleotides to prime the amplification of a target population of nucleic acid molecules, wherein each oligonucleotide comprises a hybridizing portion that consists of 6, 7, or 8 nucleotides. In some embodiments, the present invention provides a reagent comprising a population of oligonucleotides wherein the hybridizing portion is a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a reagent comprising a population of oligonucleotides that includes at least 10% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 9.9%) of the sequences set forth in SEQ ID NOS:1-933. In another embodiment, the present invention provides a reagent comprising populations of oligonucleotides wherein a defined sequence portion, such as a transcriptional promoter, (e.g., the T7 promoter (SEQ ID NO:934)) or a primer binding site (e.g., SEQ ID NO:956) is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a reagent comprising populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933.
In a further aspect, the present invention provides a kit for selectively amplifying a target population of nucleic acid molecules (e.g., all mRNA molecules expressed in a cell type except for the most highly expressed mRNA species). The kit of this aspect of the invention includes a reagent comprising a population of oligonucleotides to prime the amplification of a target population of nucleic acid molecules, wherein each oligonucleotide comprises a hybridizing portion that consists of 6, 7, or 8 nucleotides. In some embodiments, the present invention provides a kit that includes a reagent comprising a population of oligonucleotides wherein the hybridizing portion is a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a kit including a reagent that comprises a population of oligonucleotides that includes at least 10% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%) of the sequences set forth in SEQ ID NOS:1-933. In another embodiment, the present invention provides a kit that includes a reagent comprising populations of oligonucleotides wherein a defined sequence portion, such as the transcriptional promoter, (e.g., the T7 promoter (SEQ ID NO:934)), is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a kit including a reagent that comprises populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933.
In another aspect, the present invention provides methods of selectively amplifying a target population of nucleic acid molecules to generate amplified RNA molecules. The method comprises: (a) providing a population of oligonucleotides, wherein each oligonucleotide comprises a hybridizing portion and transcriptional promoter portion located 5′ to the hybridizing portion, wherein the hybridizing portion is a member of the population of oligonucleotides comprising SEQ ID NOS:1-933, (b) annealing the population of oligonucleotides to a sample comprising mRNA isolated from a mammalian subject, (c) synthesizing cDNA from the mRNA using a reverse transcriptase enzyme, (d) synthesizing double stranded cDNA using a DNA polymerase; and (e) transcribing the double-stranded cDNA into RNA using an RNA polymerase that binds to the transcriptional promoter portion of each oligonucleotide to generate amplified RNA.
In another aspect, the present invention provides methods of selectively amplifying a target population of nucleic acid molecules to generate amplified DNA molecules. The method comprises: (a) providing a first population of oligonucleotides, wherein each oligonucleotide comprises a hybridizing portion and a first PCR primer binding site located 5′ to the hybridizing portion, wherein the hybridizing portion is a member of the population of oligonucleotides comprising SEQ ID NOS:1-933, (b) annealing the population of oligonucleotides to a sample comprising mRNA isolated from a mammalian subject, (c) synthesizing cDNA from the mRNA using a reverse transcriptase enzyme, (d) synthesizing double stranded cDNA using a DNA polymerase and a second population of oligonucleotides, wherein each oligonucleotide comprises a random hybridizing portion and a second PCR binding site located 5′ to the hybridizing portion, and (e) PCR amplifying the double stranded cDNA using thermostable DNA polymerase, a first PCR primer that binds to the first PCR primer binding site and a second PCR primer that binds to the second PCR primer binding site to generate amplified double stranded DNA.
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Press, Plainsview, N.Y.; and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York, 1999, for definitions and terms of the art.
In one aspect, the present invention provides methods for selectively amplifying a target population of nucleic acid molecules. The methods of this aspect of the invention each include the step of using a population of oligonucleotides to prime the amplification (e.g., by reverse transcription, in vitro transcription, or polymerase chain reaction (PCR), or a combination thereof) of a target population of nucleic acid molecules within a larger population of nucleic acid molecules, wherein (a) each oligonucleotide comprises a hybridizing portion that consists of 6 nucleotides, or 7 nucleotides, or 8 nucleotides; and (b) the population of oligonucleotides is selected to hybridize under defined conditions to a first subpopulation of a target nucleic acid population, but not hybridize under the defined conditions to a second subpopulation of the target nucleic acid population. The population of oligonucleotides may also include a defined sequence portion located 5′ to the hybridizing portion. In one embodiment, the defined sequence portion comprises a transcriptional promoter, which can also be used as a primer binding site. Therefore, in certain embodiments of this aspect of the invention, each oligonucleotide of the population of oligonucleotides comprises a hybridizing portion that consists of 6 nucleotides, or 7 nucleotides, or 8 nucleotides; and a transcriptional promoter portion located 5′ to the hybridizing portion. In another embodiment, the defined sequence portion includes a first primer binding site for use in a PCR amplification reaction, and which may optionally include a transcriptional promoter. By way of example, the populations of oligonucleotides provided by the present invention are useful in the practice of the methods of this aspect of the invention.
For example, in one embodiment of the present invention, a population of oligonucleotides (SEQ ID NOS:1-933), that each have a length of 6 nucleotides, was identified that can be used as primers to prime the amplification of all, or substantially all, mRNA molecules from mammalian blood cells, but that do not prime the amplification of globin mRNA or ribosomal RNAs from mammalian blood cells. The identified population of oligonucleotides (SEQ ID NOS:1-933) is referred to as Not-So-Random (NSR) primers. Thus, this population of oligonucleotides (SEQ ID NOS:1-933) can be used to prime the synthesis of a population of nucleic acid molecules (e.g., cDNAs) that are representative of a starting population of mRNA molecules isolated from mammalian blood cells, but that does not include a large number of cDNA molecules that correspond to globin mRNAs or to ribosomal RNAs. The present invention also provides populations of oligonucleotides wherein a defined sequence, such as the T7 promoter (SEQ ID NO:934), is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, each oligonucleotide may include a hybridizing portion (selected from SEQ ID NOS:1-933) that hybridizes to target nucleic acid molecules (e.g., mRNAs), and a promoter sequence located 5′ to the hybridizing portion. The promoter sequence may be incorporated into DNA molecules amplified using the oligonucleotides (that include the T7 promoter) as primers, and can thereafter promote transcription from the DNA molecules. Alternatively, a defined sequence portion such as a transcriptional promoter may be covalently attached to the cDNA molecule, for example, by DNA ligase enzyme.
The first subpopulation of a target nucleic acid population can include, for example, all mRNAs expressed in a cell or tissue except for a selected group of mRNAs, such as, for example, the most abundantly expressed mRNAs. An abundantly expressed mRNA typically constitutes at least 0.1% of all the mRNA expressed in the cell or tissue (and may constitute, for example, more than 50%, or more than 60%, or more than 70% of all the mRNA expressed in the cell or tissue). An example of an abundantly expressed mRNA is globin mRNA in certain blood cells. Useful transcription promoter sequences include the T7 promoter (5′ AATTAATACGACTCACTATAGGGAGA3′) (SEQ ID NO:934)), the SP6 promoter (5′ ATTTAGGTGACACTATAGAAGNG3′ (SEQ ID NO:935)), and the T3 promoter (5′ AATTAACCCTCACTAAAGGGAGA3′ (SEQ ID NO:936)).
The methods of the invention are useful for transcriptome profiling of total RNA in a biological sample, such as whole blood, in which it is desirable to reduce the presence of a group of mRNAs (that do not hybridize to the NSR primers) from an amplified sample, such as, for example, highly expressed RNAs (e.g. globin mRNA or ribosomal RNAs). In some embodiments, the methods of the invention may be used to reduce the amount of a group of nucleic acid molecules that do not hybridize to the NSR primers in amplified nucleic acid derived from an mRNA sample by at least 2 fold up to 1000 fold, such as at least 10 fold, 50 fold, 100 fold, 500 fold, or greater, in comparison to the amount of amplified nucleic acid molecules that do hybridize to the NSR primers.
Populations of oligonucleotides used to practice the method of this aspect of the invention are selected from within a larger population of oligonucleotides, wherein (a) the subpopulation of oligonucleotides is selected based on its ability to hybridize under defined conditions to a first subpopulation of a target nucleic acid population, but not hybridize under the defined conditions to a second subpopulation of the target nucleic acid population; and (b) the population of oligonucleotides comprises all possible oligonucleotides having a length of 6 nucleotides, 7 nucleotides, or 8 nucleotides.
Composition of population of oligonucleotides. The population of oligonucleotides includes all possible oligonucleotides having a length of 6 nucleotides, or 7 nucleotides, or 8 nucleotides. The population of oligonucleotides may include only all possible oligonucleotides having a length of 6 nucleotides, or all possible oligonucleotides having a length of 7 nucleotides, or all possible oligonucleotides having a length of 8 nucleotides. Optionally the population of oligonucleotides may include other oligonucleotides in addition to all possible oligonucleotides having a length of 6 nucleotides, or all possible oligonucleotides having a length of 7 nucleotides, or all possible oligonucleotides having a length of 8 nucleotides. Typically, each member of the population of oligonucleotides is no more than 30 nucleotides long.
Sequences of population of oligonucleotides. There are 4,096 possible oligonucleotides having a length of 6 nucleotides; 16,384 possible oligonucleotides having a length of 7 nucleotides; and 65,536 possible oligonucleotides having a length of 8 nucleotides. The sequences of the oligonucleotides that constitute the population of oligonucleotides can readily be generated by a computer program, such as Microsoft Word.
Selection of subpopulation of oligonucleotides. The subpopulation of oligonucleotides is selected from the population of oligonucleotides based on the ability of the members of the subpopulation of oligonucleotides to hybridize, under defined conditions, to a first subpopulation of a target nucleic acid population, but not hybridize under the same defined conditions to a substantial number of members of a second subpopulation of the target nucleic acid population. A target nucleic acid population is a population of nucleic acid molecules (e.g., mRNA or DNA molecules) that includes nucleic acid molecules that are to be amplified (e.g., using reverse transcription, in vitro transcription, the polymerase chain reaction, or a combination thereof) to produce amplified RNA, single stranded DNA, or double stranded DNA, and also includes nucleic acid molecules that are not to be amplified. The subpopulation of oligonucleotides is made up of oligonucleotides that each hybridize, under defined conditions, to sequences distributed throughout the population of the nucleic acid molecules that are to be amplified, but that do not hybridize, under the same defined conditions, to most (or any) of the nucleic acid molecules that are not to be amplified. The subpopulation of oligonucleotides hybridizes, under defined conditions, to target nucleic acid sequences other than those that have been intentionally avoided.
For example, the population of all mRNA molecules expressed in mammalian blood cells (e.g., human blood cells) can be a target population of nucleic acid molecules. This target population contains many mRNA molecules that encode globin proteins. This target population also contains many ribosomal RNA molecules (e.g., 5S, 18S, and 28S ribosomal RNAs). It is typically undesirable to amplify the globin mRNAs or the ribosomal RNAs. For example, in gene expression experiments that analyze expression of genes in blood cells, amplification of numerous copies of abundant globin mRNAs, or ribosomal RNAs, may obscure subtle changes in the levels of less abundant mRNAs. Consequently, in the practice of the present invention, a subpopulation of oligonucleotides is selected that does not hybridize, under defined conditions, to most (or any) globin mRNAs or to most (or any) ribosomal RNAs, but that does hybridize, under the same defined conditions, to most (preferably all) of the other mRNA molecules expressed in the blood cells. Examples of blood cells include leukocytes (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes), erythrocytes, and platelets.
In order to select a subpopulation of oligonucleotides that hybridizes under defined conditions to a first subpopulation of a target nucleic acid population, but does not hybridize under the defined conditions to a second subpopulation of the target nucleic acid population, it is necessary to know the complete, or substantially complete, nucleic acid sequences of the member(s) of the second subpopulation of the target nucleic acid population. Thus, for example, if the second subpopulation includes globin mRNAs and ribosomal RNAs, it is necessary to know the nucleic acid sequences of the globin mRNAs (or a representative globin mRNA) and the 5S, 18S, and 28S ribosomal RNAs (or a representative member of each of the foregoing classes of ribosomal RNA).
A suitable software program is then used to compare the sequences of all of the oligonucleotides in the population of oligonucleotides (e.g., the population of all possible 6 nucleic acid oligonucleotides) to the sequences of the globin mRNA(s) and ribosomal RNAs to determine which of the oligonucleotides will hybridize to any portion of the globin mRNA(s) and ribosomal RNAs under defined hybridization conditions. Only the oligonucleotides that do not hybridize to any portion of the globin mRNA(s) and ribosomal RNAs, under defined hybridization conditions, are selected. Perl script may easily be written that permits comparison of nucleic acid sequences, and identification of sequences that hybridize to each other under defined hybridization conditions.
Thus, for example, as described more fully in Example 1, the subpopulation of all possible 6 nucleic acid oligonucleotides that were not exactly complementary to any portion of any ribosomal RNA sequence or that were not exactly complementary to any portion of a globin mRNA sequence were identified. In general, the subpopulation of oligonucleotides (that hybridizes under defined conditions to a first subpopulation of a target nucleic acid population, but does not hybridize under the defined conditions to a second subpopulation of the target nucleic acid population) must contain enough different oligonucleotide sequences to hybridize to all, or substantially all, nucleic acid molecules in the target nucleic acid population. Example 2 herein shows that the population of oligonucleotides having the nucleic acid sequences set forth in SEQ ID NOS:1-933 hybridizes to all, or substantially all, nucleic acid sequences within a population of gene transcripts stored in the publicly accessible database called RefSeq.
Additional defined nucleic acid sequence portions. The selected subpopulation of oligonucleotides (e.g., SEQ ID NOS:1-933) can be used to prime the amplification of a target population of nucleic acid molecules. Alternatively, a population of oligonucleotides can be used as primers wherein each oligonucleotide includes the sequence of one member of the selected subpopulation of oligonucleotides, and also includes an additional defined nucleic acid sequence. The additional defined nucleic acid sequence is typically located 5′ to the sequence of the member of the selected subpopulation of oligonucleotides. Typically, the population of oligonucleotides includes the sequences of all members of the selected subpopulation of oligonucleotides (e.g., the population of oligonucleotides can include all of the sequences set forth in SEQ ID NOS:1-933).
The additional defined nucleic acid sequence is selected so that it does not affect the hybridization specificity of the oligonucleotide to a complementary target sequence. For example, as described in
In another example, as described in
For example, one embodiment of the present invention provides a population of oligonucleotides wherein each oligonucleotide of the population includes: (a) a sequence of a 6 nucleic acid oligonucleotide that is a member of a subpopulation of oligonucleotides (SEQ ID NOS:1-933), wherein the subpopulation of oligonucleotides hybridizes to all, or substantially all, mRNAs expressed in mammalian blood cells, but does not hybridize to globin mRNAs or to ribosomal RNAs; and (b) a T7 transcriptional promoter sequence (SEQ ID NO:934) located 5′ to the sequence of the 6 nucleic acid oligonucleotide. In one embodiment, the population of oligonucleotides includes all of the 6 nucleotide sequences set forth in SEQ ID NOS:1-933. In another embodiment, the population of oligonucleotides includes at least 10% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%) of the 6 nucleotide sequences set forth in SEQ ID NOS:1-933.
Optionally, a spacer portion is located between the defined sequence portion and the hybridizing portion. The spacer portion is typically from 1 to 12 nucleotides long (e.g., from 1 to 6 nucleotides long) and can include any combination of nucleotides. The spacer portion can, for example, be composed of a random selection of nucleotides. All or part of the spacer portion may, or may not, hybridize to the same target nucleic acid sequence as the hybridizing portion. If all, or part, of the spacer portion hybridizes to the same target nucleic acid sequence as the hybridizing portion, then the effect is to enhance the efficiency of cDNA synthesis primed by the oligonucleotide that includes the hybridizing portion and the hybridizing spacer portion.
Hybridization conditions. In the practice of the present invention, a subpopulation of oligonucleotides is selected from a population of oligonucleotides based on the ability of the members of the subpopulation of oligonucleotides to hybridize, under defined conditions, to a first subpopulation of a target nucleic acid population, but not hybridize under the same defined conditions to a second subpopulation of the target nucleic acid population. The defined hybridization conditions permit the oligonucleotides to specifically hybridize to all nucleic acid molecules that are present in the sample except for globin mRNAs or ribosomal RNAs. Typically, hybridization conditions are no more than 25° C. to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex. Tm for nucleic acid molecules greater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C)−log(Na+), wherein (G+C) is the guanosine and cytosine content of the nucleic acid molecule. For oligonucleotide molecules less than 100 bases in length, exemplary hybridization conditions are 5 to 10° C. below Tm. On average, the Tm of a short oligonucleotide duplex is reduced by approximately (500/oligonucleotide length)° C. In some embodiments of the present invention, the hybridization temperature is in the range of from 40° C. to 50° C. The appropriate hybridization conditions may also be identified empirically, without undue experimentation.
Amplification Conditions. In the practice of the present invention, the amplification of the first subpopulation of a target nucleic acid population occurs under defined amplification conditions. Hybridization conditions can be chosen as described, supra. Typically, the defined amplification conditions include first strand cDNA synthesis using a reverse transcriptase enzyme. The reverse transcription reaction is performed in the presence of defined concentrations of deoxynucleoside triphosphates (dNTPs). In some embodiments, the dNTP concentration is in a range from about 1000 to about 2000 microMolar in order to enrich the amplified product for target genes, as described in Examples 5-9.
Composition and synthesis of oligonucleotides. An oligonucleotide primer useful in the practice of the present invention can be DNA, RNA, PNA, chimeric mixtures, or derivatives or modified versions thereof, so long as it is still capable of priming the desired reaction. The oligonucleotide primer can be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups or labels, so long as it is still capable of priming the desired amplification reaction.
For example, an oligonucleotide primer may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5N-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, and 2,6-diaminopurine.
Again by way of example, an oligonucleotide primer can include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
By way of further example, an oligonucleotide primer can include at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal, or analog thereof.
An oligonucleotide primer for use in the methods of the present invention may be derived by cleavage of a larger nucleic acid fragment using non-specific nucleic acid cleaving chemicals or enzymes, or site-specific restriction endonucleases, or by synthesis by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.) and standard phosphoramidite chemistry. As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209-3221, 1988) and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451, 1988).
Once the desired oligonucleotide is synthesized, it is cleaved from the solid support on which it was synthesized, and treated, by methods known in the art, to remove any protecting groups present. The oligonucleotide may then be purified by any method known in the art, including extraction and gel purification. The concentration and purity of the oligonucleotide may be determined by examining an oligonucleotide that has been separated on an acrylamide gel, or by measuring the optical density at 260 nm in a spectrophotometer.
The methods of this aspect of the invention can be used, for example, to selectively amplify coding regions of mRNAs, introns, alternatively spliced forms of a gene, and non-coding RNAs that regulate gene expression.
In another aspect, the present invention provides populations of oligonucleotides comprising at least 10% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%) of the nucleic acid sequences set forth in SEQ ID NOS:1-933. These oligonucleotides (SEQ ID NOS:1-933) can be used, for example, to prime the synthesis of cDNA molecules complementary to mRNA molecules isolated from mammalian blood without priming the synthesis of cDNA molecules complementary to globin mRNA or ribosomal RNA molecules. Indeed, these oligonucleotides (SEQ ID NOS:1-933) can be used, for example, to prime the synthesis of cDNA using any population of mRNA molecules as templates, without amplifying a significant amount of globin mRNAs or ribosomal RNAs. For example, the present invention provides populations of oligonucleotides wherein a defined sequence portion, such as a transcriptional promoter, such as the T7 promoter (SEQ ID NO:934), is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. In some embodiments, the population of oligonucleotides includes at least 10% (such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) of the 6 nucleotide sequences set forth in SEQ ID NOS:1-933.
In another aspect, the present invention provides a reagent for selectively amplifying a target population of nucleic acid molecules. The reagent can be used, for example, to prime the synthesis of cDNA molecules complementary to mRNA molecules isolated from mammalian blood cells without priming the synthesis of cDNA molecules complementary to globin mRNA or ribosomal RNA molecules. The reagent of the present invention comprises a population of oligonucleotides comprising at least 10% of the nucleic acid sequences set forth in SEQ ID NOS:1-933. In some embodiments, the present invention provides a reagent comprising a population of oligonucleotides that includes at least 10% (such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%) of the 6 nucleotide sequences set forth in SEQ ID NOS:1-933. In some embodiments, the population of oligonucleotides is selected to hybridize to substantially all nucleic acid molecules that are present in a sample except for globin mRNAs or ribosomal RNAs. In other embodiments, the population of oligonucleotides is selected to hybridize to a subset of nucleic acid molecules that are present in a sample, wherein the subset of nucleic acid molecules does not include globin mRNAs or ribosomal RNAs.
In another embodiment, the present invention provides a reagent that comprises a population of oligonucleotides wherein a defined sequence portion comprising a transcriptional promoter, such as the T7 promoter, is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a reagent comprising populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933.
In another embodiment, the present invention provides a reagent that comprises a population of oligonucleotides wherein a defined sequence portion comprising a primer binding site is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides a reagent comprising populations of oligonucleotides wherein each oligonucleotide consists of the primer binding site (SEQ ID. NO:956) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. The reagent of the present invention can be provided as an aqueous solution, or an aqueous solution with the water removed, or a lyophilized solid.
In a further embodiment, the reagent of the present invention may include one or more of the following components for the production of double-stranded cDNA: a reverse transcriptase, a DNA polymerase, a DNA ligase, a RNase H enzyme, a Tris buffer, a potassium salt, a magnesium salt, an ammonium salt, a reducing agent, deoxynucleoside triphosphates (dNTPs), [beta]-nicotinamide adenine dinucleotide (β-NAD+), and a ribonuclease inhibitor. For example, the reagent may include components optimized for first strand cDNA synthesis, such as a reverse transcriptase with reduced RNase H activity and increased thermal stability (e.g., SuperScript™ III Reverse Transcriptase, Invitrogen), and a final concentration of dNTPs in the range of from 50 to 5000 microMolar, or more preferably in the range of from 1000 to 2000 microMolar.
In another aspect, the present invention provides kits for selectively amplifying a target population of nucleic acid molecules. In some embodiments, the kits comprise a reagent that comprises a population of oligonucleotides wherein a defined sequence portion, such as a transcriptional promoter, (e.g., the T7 promoter), is located 5′ to a member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. Thus, in some embodiments, the present invention provides kits containing a reagent comprising populations of oligonucleotides wherein each oligonucleotide consists of the T7 promoter (SEQ ID NO:934) located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. The kits according to this embodiment are useful for producing in vitro transcribed RNA from cDNA generated using the not-so-random primers of the invention.
In some embodiments, the kits contain a reagent comprising populations of oligonucleotides wherein each oligonucleotide consists of a defined sequence suitable for use as a primer binding site located 5′ to a different member of the population of oligonucleotides having the sequences set forth in SEQ ID NOS:1-933. The kits according to this embodiment are useful for producing double-stranded DNA generated from PCR amplification of cDNA generated using the not-so-random primers of the invention.
The kits of the invention may be designed to detect any subpopulation of a target nucleic acid population, for example, all mRNAs expressed in a cell or tissue except for the most abundantly expressed mRNAs, in accordance with the methods described herein. Nonlimiting examples of mammalian mRNA target molecules include all or substantially all of the mRNA molecules from mammalian blood cells. Nonlimiting examples of exemplary oligonucleotide primers include SEQ ID NOS:1-933. Nonlimiting examples of the transcription promoter are set forth as SEQ ID NOS:934-936. Nonlimiting examples of primer binding regions are set forth as SEQ ID NO:946, 955, and 956.
The spacer portion may include any combination of nucleotides, including nucleotides that hybridize to the target mRNA.
In certain embodiments, the kit comprises a reagent comprising oligonucleotide primers with hybridizing portions of 6, 7, or 8 nucleotides.
In certain embodiments, the kit comprises a reagent comprising a population of oligonucleotide primers that may be used to detect a plurality of mammalian mRNA targets.
In certain embodiments, the kit comprises oligonucleotides that hybridize in the temperature range of from 40° C. to 50° C.
In another embodiment, the kit comprises a subpopulation of oligonucleotides that do not detect rRNA or globin mRNA. Exemplary oligonucleotides for use in accordance with this embodiment of the kit are provided in SEQ ID NOS:1-933.
In some embodiments, the kits comprises a reagent comprising a population of oligonucleotides comprising at least 10% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%) of the 6 nucleotide sequences set forth in SEQ ID NOS:1-933.
In certain embodiments, the kit includes oligonucleotides wherein the transcription promoter comprises the T7 promoter (SEQ ID NO:934), the SP6 promoter (SEQ ID NO:935), or the T3 promoter (SEQ ID NO:936).
In another embodiment, the kit may comprise oligonucleotides with a spacer portion of from 1 to 12 nucleotides that comprises any combination of nucleotides.
In some embodiments of the present invention, the kit may further comprise one or more of the following components for the production of cDNA—a reverse transcriptase enzyme, a DNA polymerase enzyme, a DNA ligase enzyme, a RNase H enzyme, a Tris buffer, a potassium salt (e.g., potassium chloride), a magnesium salt (e.g., magnesium chloride), an ammonium salt (e.g., ammonium sulfate), a reducing agent (e.g., dithiothreitol), deoxynucleoside triphosphates (dNTPs), [beta]-nicotinamide adenine dinucleotide (β-NAD+), and a ribonuclease inhibitor. For example, the kit may include components optimized for first strand cDNA synthesis, such as a reverse transcriptase with reduced RNase H activity and increased thermal stability (e.g., SuperScript™ III Reverse Transcriptase, Invitrogen), and a dNTP stock solution to provide a final concentration of dNTPs in the range of from 50 to 5,000 microMolar, or more preferably in the range of from 1000 to 2000 microMolar.
In various embodiments, the kit may include a detection reagent such as SYBR green dye or BEBO dye that preferentially or exclusively binds to double stranded DNA during a PCR amplification step. In other embodiments, the kit may include a forward and/or reverse primer that includes a fluorophore and quencher to measure the amount of the PCR amplification products.
A kit of the invention can also provide reagents for in vitro transcription of the amplified cDNAs. For example, in some embodiments, the kit may further include one or more of the following components—a RNA polymerase enzyme, an IPPase (Inositol polyphosphate 1-phosphatase) enzyme, a transcription buffer, a Tris buffer, a sodium salt (e.g., sodium chloride), a magnesium salt (e.g., magnesium chloride), spermidine, a reducing agent (e.g., dithiothreitol), nucleoside triphosphates (ATP, CTP, GTP, UTP), and amino-allyl-UTP.
In another embodiment, the kit may include reagents for labeling the in vitro transcription products with Cy3 or Cy5 dye for use in hybridizing the labeled cDNA samples to microarrays.
The kit optionally includes instructions for using the kit in the selective amplification of mRNA targets. The kit can also be optionally provided with instructions for in vitro transcription of the amplified cDNA molecules, and with instructions for labeling and hybridizing the in vitro transcription products to microarrays.
In another aspect, the present invention provides methods of selectively amplifying a target population of nucleic acid molecules to generate amplified RNA molecules. The method comprises: (a) providing a population of oligonucleotides, wherein each oligonucleotide comprises a hybridizing portion and transcriptional promoter portion located 5′ to the hybridizing portion, wherein the hybridizing portion is a member of the population of oligonucleotides comprising SEQ ID NOS:1-933, (b) annealing the population of oligonucleotides to a sample comprising mRNA isolated from a mammalian subject, (c) synthesizing cDNA from the mRNA using a reverse transcriptase enzyme, (d) synthesizing double stranded cDNA using a DNA polymerase; and (e) transcribing the double-stranded cDNA into RNA using an RNA polymerase that binds to the transcriptional promoter portion of each oligonucleotide to generate amplified RNA.
In another aspect, the present invention provides methods of selectively amplifying a target population of nucleic acid molecules to generate amplified DNA molecules. The method comprises: (a) providing a first population of oligonucleotides, wherein each oligonucleotide comprises a hybridizing portion and a first PCR primer binding site located 5′ to the hybridizing portion, wherein the hybridizing portion is a member of the population of oligonucleotides comprising SEQ ID NOS:1-933, (b) annealing the population of oligonucleotides to a sample comprising mRNA isolated from a mammalian subject, (c) synthesizing cDNA from the mRNA using a reverse transcriptase enzyme, (d) synthesizing double stranded cDNA using a DNA polymerase a second population of oligonucleotides, wherein each oligonucleotide comprises a random hybridizing portion and a second PCR binding site located 5′ to the hybridizing portion, and (e) PCR amplifying the double stranded cDNA using thermostable DNA polymerase, a first PCR primer that binds to the first PCR primer binding site and a second PCR primer that binds to the second PCR primer binding site to generate amplified double stranded DNA.
In accordance with this aspect of the invention, any DNA-dependent DNA polymerase may be utilized to synthesize second-strand DNA molecules from the first strand cDNA. For example, the Klenow fragment of DNA Polymerase I can be utilized to synthesize the second-strand DNA molecules. The synthesis of second-strand DNA molecules is primed using a second population of oligonucleotides comprising a hybridizing portion consisting of from 6 to 9 random nucleotides and further comprising a defined sequence portion 5′ to the hybridizing portion. The defined sequence portion may include any suitable sequence, provided that the sequence differs from the defined sequence contained in the first population of oligonucleotides. Depending on the choice of primer sequence, these defined sequence portions can be used, for example, to selectively direct DNA-dependent RNA synthesis from the second DNA molecule and/or to amplify the double-stranded cDNA template via DNA-dependent DNA synthesis.
Purification of double-stranded DNA molecules: Synthesis of the second DNA molecules yields a population of double-stranded DNA molecules wherein the first DNA molecules are hybridized to the second DNA molecules, as shown in
Amplification of the double-stranded DNA molecules. In the practice of the methods of this aspect of the invention, the double-stranded DNA molecules are utilized as templates that are enzymatically amplified using the polymerase chain reaction. Any suitable primers can be used to prime the polymerase chain reaction. Typically two primers are used, one primer hybridizes to the defined portion of the first primer sequence (or to the complement thereof); and the other primer hybridizes to the defined portion of the second primer sequence (or to the complement thereof).
PCR Amplification Conditions. In general, the greater the number of amplification cycles during the polymerase chain reaction, the greater the amount of amplified DNA that is obtained. On the other hand, too many amplification cycles may result in randomly biased amplification of the double-stranded DNA. Thus, in some embodiments, a desirable number of amplification cycles is between 5 and 40 amplification cycles, such as from five to 35, such as from 10 to 30 amplification cycles.
With regard to temperature conditions, typically a cycle comprises a melting temperature, such as 95° C.; an annealing temperature, which varies from about 40° C. to 70° C.; and an elongation temperature, which is typically about 72° C. With regard to the annealing temperature, in some embodiments the annealing temperature is from about 55° C. to 65° C., more preferably about 60° C.
In one embodiment, amplification conditions for use in this aspect of the invention comprise 10 cycles of (95° C., 30 sec; 60° C., 30 sec; 72° C., 60 sec), then 20 cycles of (95° C., 30 sec; 60° C., 30 sec, 72° C., 60 sec (+10 sec added to the elongation step with each cycle)).
With regard to PCR reaction components for use in the methods of this aspect of the invention, dNTPs are typically present in the reaction in a range from 50 μM to 2000 μM dNTPs, and more preferably from 800 to 1000 μM. MgCl2 is typically present in the reaction in a range from 0.25 mM to 10 mM, and more preferably about 4 mM. The forward and reverse PCR primers are typically present in the reaction from about 50 nM to 2000 nM, and more preferably present at a concentration of about 1000 nM.
DNA Labeling. Optionally, the amplified DNA molecules can be labeled with a dye molecule to facilitate use as a probe in a hybridization experiment, such as a probe used to screen a DNA chip. Any suitable dye molecules can be utilized, such as fluorophores and chemiluminescers. An exemplary method for attaching the dye molecules to the amplified DNA molecules is provided in EXAMPLE 15.
The methods according this aspect of the invention may be used, for example, for transcriptome profiling in a biological sample containing total RNA, such as whole blood. In some embodiments, the amplified DNA produced in accordance with the methods of this aspect of the invention is labeled for use in gene expression experiments, thereby providing a hybridization based reagent that typically produces a lower level of background than amplified RNA generated from NSR-primed cDNA.
In some embodiments of this aspect of the invention, the defined sequence portion of the first and/or second primer binding regions further includes one or more restriction enzyme sites, thereby generating a population of amplified double-stranded DNA products having one or more restriction enzyme sites flanking the amplified portions. These amplified products may be used directly for sequence analysis, or may be released by digestion with restriction enzymes and subcloned into any desired vector, such as an expression vector for further analysis.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.
This Example describes the selection of a population of 933 6-mer oligonucleotides (SEQ ID NOS:1-933) that hybridizes to all, or substantially all, mRNA molecules expressed in blood cells, but that does not hybridize to globin mRNA or to ribosomal RNA.
All 4096 possible 6-mer oligonucleotides were computed, wherein each nucleotide was A, T (or U), C, or G. The reverse complement of each 6-mer oligonucleotide was compared to the nucleotide sequences of 18S and 28S rRNAs, and to the nucleotide sequences of the following six hemoglobin genes, selected based on their high level of expression in blood samples:
Reverse-complement 6-mer oligonucleotides having perfect matches to any of the eight transcript sequences were eliminated. The reverse complements of 933 6-mers (SEQ ID NOS:1-933) did not perfectly match any portion of the globin or rRNA transcripts. The 933 6-mer oligonucleotides (SEQ ID NOS:1-933) that do not have a perfect match to any portion of the globin or rRNA genes are referred to as “Not-So-Random” (“NSR”) primers.
This Example shows that the population of oligonucleotides having the nucleic acid sequences set forth in SEQ ID NOS:1-933 hybridize to every 4 to 5 nucleotides on a nucleic acid sequence within the RefSeq database accessible at the website of the National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, Md. 20894, U.S.A. NCBI's reference sequence transcript database (RefSeq) contains what is considered a gold-standard of human protein coding transcripts.
Random 6-mers (N6) can anneal at every nucleotide position on a transcript sequence from the RefSeq database (represented as “nucleotide sequence”), as shown in
Thus, the population of 933 6-mers (SEQ ID NOS:1-933) is capable of amplifying all transcripts except 18S, 28S, and hemoglobin transcripts.
This Example shows that PCR amplification of an actin reporter mRNA using the 933 6-mers (SEQ ID NOS:1-933) (that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end) selectively reduces priming of globin mRNA and rRNA.
Total RNA was extracted from individual donors of human whole blood using the PAXgene Blood RNA Kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's instructions. MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (Epicentre Biotechnologies, Madison, Wis.) was used to synthesize cDNA from 100 ng of template RNA with 5 μM 6-mers (SEQ ID NOS:1-933) (T7-NSR6) or random 9-mers with the T7 promoter covalently attached at the 5′ end (T7-N9). Prior to reverse transcription, 5 μL of water containing primers and template were denatured at 65° C. for 5 min, snap cooled at 4° C. and equilibrated to 23° C. For reverse transcription, 5 μL of RT master mix containing 1 μl of water, 2 μl of 5× First Strand Buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2; Invitrogen Corporation, Carlsbad, Calif.), 0.5 μl of 100 mM DTT, 0.5 μl of 10 mM dNTPs and 1.0 μl of MMLV reverse transcriptase (50 units/μl) was added to the sample mix. The 10 μl reaction was incubated at 40° C. for 120 min, 95° C. for 5 min, cooled to room temperature, and diluted 5-fold with water.
Following reverse transcription, duplicate measurements of 2 μl of cDNA were made in 10 μl final reaction volumes by quantitative PCR (qPCR) in a 384-well optical PCR plate using a 7900 HT PCR instrument (Applied Biosystems, Foster City, Calif.). qPCR was performed using ABI TaqMan® assays (eukaryotic 18S rRNA, ABI Catalog Hs99999901 s1; human beta actin, ABI Catalog No. 4310881E; human beta hemoglobin, ABI Catalog No. Hs00747223_g1; ABI Assay-by-Design alpha globin; (fwd 5′-GCACGCGCACAAGCT-3′ (SEQ ID NO:937), reverse 5′-GGGTCACCAGCAGGCA-3′ (SEQ ID NO:938), FAM probe 5′-ACTTCAAGCTCCTAAGCCAC-3′ (SEQ ID NO:939)) using the manufacturer's recommended conditions. Following PCR, the results table was exported to Excel (Microsoft Corp., Redmond, Wash.) and quantitative analysis for samples was regressed from the raw data (abundance=10[(Ct-5)/-3.4]).
Reverse transcription of human whole blood total RNA with the 933 NSR 6-mers (SEQ ID NOS:1-933) resulted in a significant reduction of globin and rRNA content in cDNA as determined by qPCR. Specifically, T7-NSR 6 primed cDNA showed reduced abundance of alpha globin (58% reduction), beta globin (85% reduction) and 18S rRNA (75% reduction) as compared to cDNA generated with random 9-mers (T7-N9).
As shown below in TABLE 2, T7-NSR6 primed cDNA showed reduced abundance of beta globin (HBB). (94% reduction), and 18S rRNA (91% reduction), as compared to cDNA generated with random 9-mers. In contrast, beta actin cDNA levels for the two primer pools (T7-N9 and T7-NSR6) were comparable after amplification of RNA, a result that was confirmed using three independent donors (data not shown).
This Example shows that the 933 6-mers (SEQ ID NOS:1-933) (that each have the T7 promoter (SEQ ID NO:934) covalently attached at the ˜5′ end) prime the amplification of a substantial fraction of the transcriptome.
To assess cDNA complexity, 933 6-mers (SEQ ID NOS:1-933) were used to amplify total RNA from two diverse cell lines (Jurkat and K562) using a two-step reverse transcription and in vitro transcription (IVT) approach. The amplification products were labeled and hybridized to a DNA microarray containing probes for ˜5000 human transcripts known to be expressed in either Jurkat or K562 cells. Nearly 82% (4244/5198) of the transcripts reported above background signal intensity (log intensity ≧−1.5) in either cell line. Moreover, the Jurkat/K562 expression profile from RNA amplified using the 933 6-mers (SEQ ID NOS:1-933) showed a high degree of similarity to a control profile amplified from random-primed Jurkat and K562 mRNA (for probes with a p-value <0.01 in either experiment; correlation coefficient=0.72).
The amplification experiment was conducted as follows. Total RNA and mRNA were obtained from Ambion, Inc. (Austin, Tex.) for the cell lines Jurkat (T lymphocyte, ATCC No. TIB-152) and K562 (chronic myelogenous leukemia, ATCC No. CCL-243). A two-step amplification approach using reverse transcription and in vitro transcription was used to generate amplified RNA (aRNA) for microarray hybridizations. NSR cDNA was synthesized from 1 μg of total RNA and 5 μM primer in a 20 μL reaction volume as described in Example 3, but with a prolonged incubation step at 40° C. for 6 hrs. cDNA was synthesized from 100 ng of mRNA using 10 μM random 9-mer in a 20 μL reaction volume as described in Example 3. The 933 6-mers (SEQ ID NOS:1-933) and the random 9-mers were covalently linked to the T7 promoter sequence (SEQ ID NO:934) at the 5′ end.
For in vitro transcription, the 20 μL RT reaction was added to 60 μL of IVT pre-mix containing 16 μL of 5× Transcription Buffer (0.2 M Tris-HCl, pH 7.5, 50 mM NaCl, 30 mM MgCl2, and 10 mM spermidine; Epicentre Biotechnologies, Madison, Wis.), 6 μL of 100 mM DTT, 3.3 μL of 200 mM MgCl2, 8 μL of NTP (25 mM ATP, 25 mM CTP, 25 mM GTP, 6 mM UTP), 2 μL of 75 mM amino allyl-UTP, 0.6 μL of IPPase (2 U/μL), 0.08 μL, of T7 RNA polymerase (2.5 kU/μL), 24 μL of water and incubated for 16 hrs at 40° C., 5 min at 70° C., and cooled to room temperature. The final product was coupled to Cy3 or Cy5 dye in 1 M bicarbonate buffer for 1 h. Reactions were finished with the addition of 4 M hydroxylamine followed by purification with RNAeasy Spin Columns. The percentage dye incorporation and total cDNA yield were determined spectrophotometrically. Pairs of Cy3/Cy5-labeled cDNA samples were combined and hybridized to microarrays as described previously (Roberts et al., “Signaling and Circuitry of Multiple MAPK Pathways Revealed by a Matrix of Global Gene Expression Profiles,” Science 287:873-880, 2000). Arrays were hybridized for 48 hrs then washed and scanned on Agilent Microarray Scanners.
Custom high density oligonucleotide arrays were obtained from Agilent Technologies (Palo Alto, Calif.). A total of 5198 transcripts were chosen for array probe design from previously described experiments (Hughes et al., “Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer,” Nat. Biotechnol. 19(4):342-347, 2001). Probe sequences (60-mers) were selected on the basis of nucleotide composition and cross-hybridization potential, and positioned at least 500 bp upstream of the known polyadenylation site for each transcript. Data analysis was carried out as previously described (Hughes et al., supra).
This example shows that increasing the concentration of deoxyribonucleoside triphosphates (dNTPs) in the first strand synthesis reaction increases the amplification of IGF1R and GAPDH reporter mRNA using NSR primers (comprising SEQ ID NOS:1-933) (that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end), relative to globin mRNA and rRNA.
Methods:
NSR primers were used in this Example as follows:
The 933 6-mers (SEQ ID NOS:1-933) plus a single random nucleotide inserted between the 6-mer sequence and the T7 promoter (SEQ ID NO:934), referred to as “NSR7.”
Total RNA was extracted from human whole blood as described in Example 3. MMLV reverse transcriptase (SuperScript III™ (“SSIII”), Invitrogen Corporation, Carlsbad, Calif.) was used to synthesize cDNA from 200 ng of template RNA with 10 μM 6-mers (SEQ ID NOS:1-933) or random 8-mers (N8). First strand synthesis was performed as described in Example 3, except that the reaction volume was 20 μL, 2 μL SSIII reverse transcriptase (200 units/μl) was added to the reaction mix, and the concentration of dNTPs varied from 500 to 5,000 μM. The reaction was incubated at 40° C. for 120 min, 70° C. for 15 min, cooled to room temperature, and diluted two-fold with 10 mM Tris pH 7.6, 0.1 mM EDTA.
Following reverse transcription, qPCR was performed as described in Example 3, except that ABI TaqMan assays for human insulin growth factor one receptor (IGFR1R) (ABI Catalog #Hs00181385) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ABI Catalog #43100884E-0507028) were substituted for human beta actin as reporters. Following PCR, the results table was exported to Excel and quantitative analysis for samples was regressed from the raw data as described in Example 3.
Reverse transcription of human whole blood total RNA with the NSR7 primers (comprising SEQ ID NOS:1-933) and increasing concentrations of dNTPs resulted in increased specificity of the NSR7-primed reaction. The data from two independent experiments is shown in Tables 1-5 below. The raw abundance values, expressed as 10 raised to the (Cts×log 10(½)+10) power (Tables 3 and 4), were normalized to the highest N8 value for a given target gene (Tables 5 and 6).
The ratios of reporters to backgrounds (reporter/background), which is the ratio of IGF1R and GAPDH reporters to 18S rRNA, 28S rRNA, alpha globin (HBA) and beta globin (HBB) background values, were then calculated for the NSR7 and N8 primer pools. As shown in Tables 7 and 8, the NSR7-primed reporter/background ratio increased dramatically with increasing dNTP concentrations.
The ratios of ratios is a comparison of reporter to background ratios for the NSR7 and N8 primer pools, and measures the specificity of the NSR-primed reactions. As shown in Table 9 and Table 10, the ratios of ratios increased dramatically at higher dNTP concentrations.
The yield ratio measures the output of cDNA conversion primed by NSR7 versus N8, expressed as a percentage (NSR divided by N8). As shown in Table 11 and Table 12, the NSR to N8 yield ratio was substantially higher for the IGF1R and GAPDH reporters than for the rRNA and globin mRNA. The ratio was relatively constant across dNTP concentrations for the IGF1R and GAPDH reporters, but decreased with increasing dNTP concentrations for the rRNA and globin genes, consistent with enrichment of reporters at higher dNTP concentrations.
In summary, this example shows that the absolute yields of cDNA primed with the NSR7 primers (comprising SEQ ID NOS:1-933) were highest at low dNTP concentrations, whereas the specificity relative to random 8-mers increased at higher dNTP concentrations. Based on this data, it appears that the optimal concentration of dNTPs to achieve high yield and high specificity is in the range of about 1000 to about 2000 μM dNTPs.
This Example shows that NSR primers (comprising SEQ ID NOS:1-933), that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end, maintain specificity when low amounts of template RNA is reverse transcribed and amplified by in vitro transcription (WT).
In initial experiments, it was determined that the NSR7-primed specificity was lost during the second strand synthesis and IVT amplification steps when low amounts of template RNA were used. The IVT amplification step is required to produce enough RNA for the microarray experiments disclosed above.
It was observed that the NSR7-primed specificity from low amounts of template RNA was improved by using the SuperScript™ RNA Amplification System (Invitrogen, Catalog No. L1016-01), as follows:
Methods: First strand cDNA synthesis was performed as described in Example 5 using 1000 μM dNTPs, and 50 ng, 100 ng, and 200 ng of whole blood total RNA template with the NSR7 primer pool. 100 ng of RNA template was used with random 7-mers that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end (T7-N7) and random 8-mer (N8) primers. For second strand cDNA synthesis, 20 μL of the first strand reaction product was added to 130 μL of second strand mix containing 91 μL water, 30 μL 5× Second-Strand Reaction Buffer (100 mM Tris-HCl (pH 6.9), 450 mM KCl, 23 mM MgCl2, 0.75 mM β-NAD+, 50 mM (NH4)2SO4) 3 μL 10 mM dNTPs, 4 μL DNA Polymerase I (10 units/μl), 1 μL DNA ligase (10 units/μl) and 1 μL RNase H (2 units/μl). The reaction was incubated at 16° C. for 120 min. The double stranded cDNA was purified using the Spin Cartridge and buffers supplied in the kit (Invitrogen), and the volume of the eluted cDNA was reduced to less than 20 μL by centrifugation under vacuum with low to moderate heat.
The double stranded cDNA template was amplified by in vitro transcription using T7 polymerase to transcribe antisense RNA (aRNA) complementary to the original mRNA targets. For IVT, the volume of the purified cDNA was adjusted to 23 μL with water, added to 17 μL of IVT reaction mix containing 4 μL 10×T7 Reaction Buffer (Invitrogen), 7 μL of T7 Enzyme Mix (includes T7 RNA polymerase), 8 μL NTP (25 mM each of ATP, CTP, GTP, and UTP), and incubated at 37° C. for 6 to 16 hours. The resulting amplified aRNA was purified using the Spin Cartridges and aRNA Binding and Wash Buffers supplied by Invitrogen, and the yield of aRNA was quantified using Nanodrop Technologies.
The aRNA was reverse transcribed with random 8-mers (N8) to produce single stranded cDNA. For reverse transcription, 500 ng or 1000 ng of aRNA was added to 20 μl of RT mix containing 4 μl 5× first strand buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2), 2 μl 10 mM dNTPs, 1 μl 100 mM DTT, 1 μl RNAseOUT™ (40 units/μl), 2 μl SuperScript III Reverse Transcriptase (200 units/μl), and incubated at 40° C. for 60 min, 70° C. for 10 min, and cooled on ice.
Following reverse transcription, qPCR was performed as described in Example 3 using ABI Taqman® assays for IGFR1 (catalog #Hs00181385), GAPDH (catalog #43100884E-0507028), GUSB (catalog #4310888E), ACTIN (catalog #4310881E), eukaryotic 18SrRNA (catalog #Hs99999901s1), and human hemoglobin beta (HBB) (catalog #Hs00747223 g1)
For eukaryotic 28S rRNA and human hemoglobin alpha (HBA) custom primers and probes (containing a 5′-FAM (6-carboxyfluorescein) reporter dye and a no fluorescent quencher (NFQ) at the 3′ end of the probe) were used as follows:
Reverse transcription of human whole blood total RNA with the NSR7 (comprising SEQ ID NOS:1-933) followed by IVT produced high yields of aRNA that were directly proportional to the amount of input RNA template as shown in
Quantitative PCR (qPCR) analysis of the cDNA and reverse transcribed IVT products (IVT-RT) from varying amounts of RNA template is shown in Table 13 (raw Ct values) and Table 14 (absolute abundance). Primer pool indicates the primers used for first strand cDNA synthesis. Product indicates either the cDNA or IVT-RT product was the template for qPCR.
The ratio of reporter to background RNA is shown in Table 15. There was little substantial NSR7 specificity relative to T7-N7 observed during the initial first strand cDNA step (left columns). Importantly, the ratio of reporter to background RNAs increased going from the first strand synthesis step to the IVT step (right columns) for both the NSR7 and T7-N7 primers (the data was normalized to N8).
Quantitative PCR analysis of the IVT products generated from NSR7-primed cDNA showed that the composition of each sample was approximately the same at three different total RNA input concentrations (50 ng, 100 ng, and 200 ng). Composition refers to normalized yields of reporter and background IVT products. As shown in Table 16, the normalized yields were very similar for both the reporter and background genes across a 4-fold range of input RNA. This data suggests that the IVT products were amplified from the appropriate target RNA and the reaction was not overwhelmed with nonspecific transcript material.
This Example shows that NSR7 primers (comprising SEQ ID NOS:1-933), that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end, coupled with high dNTP concentrations, generate first strand RT products with high specificity, and that these products can be amplified with the SuperScript™ RNA Amplification System (Invitrogen) to produce high yields of IVT product substantially enriched for target genes.
Methods:
400 ng of whole blood total RNA was reverse transcribed using the NSR7, T7-N7, and N8 primers in 401 reaction volume, as described in Example 5. 20 μl (200 ng whole blood total RNA) was used to assess IGF1R, GAPDH, rRNA and globin abundance by qPCR, and the best performing set of primers was then subjected to IVT. Two primer concentrations (10 μM and 25 μM) and two dNTP concentrations (1000 μM and 2500 μM) were evaluated. The raw Ct and absolute abundance values following first strand reverse transcription are shown in Table 17 and Table 18.
One μg of aRNA from the IVT reaction was converted back into cDNA by reverse transcription for qPCR analysis, as described in Example 6.
The raw Ct and abundance values following qPCR of the IVT-RT reaction products is shown in Tables 19 and 20. The reporter to background ratios for first strand NSR7 and IVT-RT are shown in Table 21.
The ratio of ratios data allows a comparison of first strand synthesis yields with IVT yields following conversion to cDNA and qPCR. Comparison of reporter to background ratios and NSR7 to N8 ratios shows that increasing the dNTP concentration from 1000 mM to 2500 mM resulted in a pronounced increase in NSR7-mediated specificity, as shown in Table 22. The primer concentration had a less noticeable impact on the NSR7-mediated specificity. In addition, comparing the ratios of NSR7-primed cDNA synthesis yields to the IVT yields showed a dramatic relative reduction in rRNAs (18S and 28S), suggesting that the rRNA cDNAs observed in the first round of reverse transcription fail to be transcribed during IVT (Table 23). The relative reduction in background globin mRNA yields between first strand synthesis and IVT was not observed with the NSR7 primers. When first strand synthesis was primed with random 8-mers, the reporter genes IGF1R and GAPDH were enriched relative to both rRNA and globin mRNA following IVT (Table 24).
In summary, this Example shows that high dNTP concentrations in combination with the NSR7 primers (comprising SEQ ID NOS:1-933) generate first strand reverse transcription products with high NSR7 specificity. The first strand products can then be amplified using the SuperScript™ RNA Amplification System (Invitrogen) to produce high yields of IVT product that is substantially enriched for target genes.
This Example shows that selective enhancement of IGF1R and GAPDH reporter mRNAs relative to rRNA and globin mRNA using the NSR7 primers (comprising SEQ ID NOS:1-933), that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end, with high concentrations of dNTPs occurs over a wide range of whole blood total RNA template concentrations.
Total human whole blood RNA was reverse transcribed and analyzed by qPCR as described in Example 5. The amount of template RNA was 20 ng, 50 ng, 100 ng, 500 ng, 1000 ng, 2000 ng, and 5000 ng. The data show that the NSR7 mediated enrichment of IGF1R and GAPDH reporters relative to rRNA and globin mRNA (as determined by the ratio of ratios for NSR7 versus N8) is observed at all template amounts (Table 25 and Table 26). The reporter/background ratio of ratios for NSR versus N8 increases at higher template amounts, but the overall yield of first strand cDNA product is compromised (Table 27). One microgram (1000 ng) of input template RNA represents an optimal compromise between cDNA yields and NSR7-mediated amplification specificity. At higher RNA template amounts (2000 ng and 5000 ng) the reverse transcription reaction failed to fully convert RNA into cDNA. The cDNA yields in Table 28 were determined by multiplying the absolute yields of product (from Table 29) by the dilution factor relative to 5000 ng of template RNA, then normalizing to the N8 yield from 20 ng of template RNA which consistently produced the highest yield values. The normalized cDNA yields of IGF1R and GAPDH were similar across all template amounts for both NSR7 and N8, with a decreased yield at the highest RNA template concentrations. In contrast, the yield of 18S, 28S, alpha globin, and beta globin products was substantially higher when primed with N8 than when primed with NSR7.
In summary, the “NSR effect,” defined as a high percentage of conversion by the NSR7 primers (comprising SEQ ID NOS:1-933) of reporter mRNA to cDNA relative to N8, but a low percentage of conversion by the NSR7 primers of background RNAs to cDNA relative to N8, is conserved across all template concentrations tested. This provides the advantage that reaction conditions do not need to be customized to template amounts.
This Example shows that the NSR7 primers (comprising SEQ ID NOS:1-933), that each have the T7 promoter (SEQ ID NO:934) covalently attached at the 5′ end, combined with high dNTP concentrations show increased specificity when compared to random 7-mers (T7-N7), and that this specificity is maintained across a range of RNA template amounts.
First-strand cDNA synthesis, second-strand synthesis, IVT, reverse transcription of the IVT, and qPCR of the aRNA were performed as described in Example 6. The RNA template amounts were 100 ng, 200 ng, 500 ng, and 1000 ng of whole blood total RNA. The reaction volumes were 40 μl, and the reactions were incubated at 40° C. for 180 min, 70° C. for 10 min, and chilled on ice. The dNTP concentration was 2000 μM. Second strand synthesis reaction was for 120 min at 16° C.
The data from the first-strand synthesis indicated that high dNTP concentrations result in increased levels of IGF1R and GAPDH reporters relative to rRNA and globin background genes when primed with the NSR7 pool, but not when primed with the T7-N7 pool (data normalized to N8) (Table 30). See
As shown in
In summary, high dNTP concentrations in combination with the NSR primers result in increased reporter levels and decreased background levels following first strand cDNA synthesis and IVT across a range of RNA template amounts. The composition of the NSR-primed IVT product is substantially different than the T7-N7 and N8 primed product, such that IGF1R and GAPDH levels increased while rRNA and globin levels decreased when primed with NSR, but were not substantially different than the N8-primed starting material when primed with T7-N7. Finally, the composition of the NSR-primed IVT product was relatively uniform across a range of RNA template amounts.
This Example describes an exemplary method of using NSR-primed first strand reverse transcription, followed by second strand synthesis and in vitro transcription for the preparation of microarray samples.
Methods:
First Strand Reverse Transcription
First Strand Reverse Transcription is carried out as follows:
Combine:
Add 10 μl of RT cocktail (prepared on ice) containing:
The sample is mixed, centrifuged and transferred to a 40° C. pre-warmed thermal cycler (to provide a “hot start”), and the sample is incubated at 40° C. for three hours, 70° C. for 10 minutes and chilled to 4° C.
Second Strand Synthesis
A second strand synthesis cocktail is prepared using the components of the SuperScript™ RNA Amplification System (Invitrogen, Catalog No. L1016-01) as follows:
130 μl of the second strand synthesis cocktail is added to the 20 μl first strand reaction described above, mixed and the sample is incubated at 16° C. for two hours.
Purification of Double-Stranded cDNA
1. 500 μl of cDNA Loading Buffer is added to the reaction tube from Second-Strand cDNA Synthesis. The total volume in the tube should be 650 μl. Mix thoroughly by pipetting up and down.
2. Each Spin Cartridge is pre-inserted into a collection tube. Load the cDNA/buffer solution directly onto the Spin Cartridge.
3. Centrifuge at 12,000×g at room temperature in a microcentrifuge for 1 minute. Remove the collection tube and discard the flow-through.
4. Place the Spin Cartridge in the same collection tube and add 700 μl of cDNA Wash Buffer.
5. Centrifuge at 12,000×g at room temperature for 2 minutes. Remove the collection tube and discard the flow-through.
6. Place the Spin Cartridge in the same collection tube and centrifuge at 12,000×g at room temperature for an additional 4 minutes. Remove the collection tube and discard the flow-through.
7. Place the Spin Cartridge into a new Recovery Tube.
8. Add 28 μl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 2 minutes.
9. Centrifuge at 12,000×g at room temperature for 1 minute.
10. Pipette cDNA back onto column, let sit 1 min, and recentrifuge.
In Vitro Transcription Reaction
An In Vitro Transcription Cocktail is prepared as follows (18 μl volume)
22 μl of purified cDNA is added to the cocktail, and the sample is incubated at 37° C. overnight.
Purification of aRNA
The following procedure is used to purify the aRNA.
1. Add 160 μl of aRNA Binding Buffer to the reaction tube. The total volume should be 200 μl. Mix thoroughly by pipetting up and down.
2. Add 100 μl of 100% ethanol to the reaction tube. Mix thoroughly by pipetting up and down.
3. Each Spin Cartridge is pre-inserted into a collection tube. Load the entire aRNA/buffer solution directly onto the Spin Cartridge.
4. Centrifuge at 12,000×g in a microcentrifuge for 15 seconds at room temperature. Remove the collection tube and discard the flow-through.
5. Place the Spin Cartridge in the same collection tube and add 500 μl of aRNA Wash Buffer.
6. Centrifuge at 12,000×g for 15 seconds at room temperature. Remove the collection tube and discard the flow-through.
7. Repeat Steps 5-6.
8. Place the Spin Cartridge in the same collection tube and centrifuge at full speed for an additional 2 minutes to dry the column. Remove the collection tube and discard the flow-through.
9. Place the Spin Cartridge into a new Recovery Tube.
10. Add 100 μl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.
11. Centrifuge at 12,000×g for 2 minutes at room temperature to collect the purified aRNA in the eluate.
12. Quantify aRNA yield using Nanodrop.
This Example shows that cDNA generated using the 933 6-mers (SEQ ID NOS:1-933 either with or without T7 promoter (SEQ ID NO:934) covalently attached) can be converted into aDNA by PCR.
Methods:
NSR primers used in this Example were as follows:
The 933 6-mers (SEQ ID NOS:1-933) plus a single random nucleotide inserted between the 6-mer sequence and the T7 promoter (SEQ ID NO:934), referred to as “NSR7.”
Total RNA for the cell line Jurkat (T lymphocyte, ATCC No. TIB-152) was obtained from Ambion, Inc. (Austin, Tex.).
The protocol involves a three-step amplification approach. (1) first strand cDNA is generated from RNA using reverse transcription that is primed with NSR primers comprising a first primer binding site to generate NSR primed first strand cDNA, (2) second strand cDNA synthesis that is primed with random primers (e.g., 9-mers) comprising a second primer binding site; and (3) PCR amplification of the cDNA that is primed with forward and reverse primers that bind to the first and second primer binding sites to generate amplified DNA (aDNA).
Note: the protocol described below was carried out with all the primers shown above in TABLE 35, however it is briefly described with reference to Primer Set 1 in order to clearly present the three method steps:
First Strand cDNA synthesis: was primed with the NSR7 primer pool comprising the T7 promoter (SEQ ID NO:934) plus one random nucleotide (N) plus the 933 6-mers (SEQ ID NOS:1-933)
Second Strand cDNA synthesis: was carried out using the following primer pool:
5′ TGCATTGAGAGGGTGTAATTTGNNNNN 3′ (SEQ ID NO:947) (referred to as R RT 1 comprising random 9-mers plus second PCR primer binding site, or “tail”).
PCR Amplification was primed using the following PCR Primers:
Forward PCR 1: 5′ CGCAATTAATACGACTCACTATAGG 3′ (SEQ ID NO:946) (binds to T7 promoter (first primer binding site))
Reverse PCR 1: 5′ TGCATTGAGAGGGTGTAATTTG 3′ (SEQ ID NO:947)
(binds to second PCR primer binding site)
Additional Primer Sets Useful for Generating aDNA:
Although the primer sets described above in Table 35 are used with NSR7 including the T7 promoter, it will be understood that the method of producing aDNA may be practiced using a pool of not-so-random primers (NSR) tailed with any defined sequence containing at least one primer binding sequence. For example, a primer pool was used comprising a pool of NSR primers that included the 933 6-mers (SEQ ID NOS:1-933) plus a single random nucleotide inserted between the 6-mer sequence and the first primer binding sequence, SEQ ID NO:956: 5′ CCGAACTACCCACTTGCATT 3′
First strand synthesis was carried out using the NSR primer pool comprising the first primer binding sequence (SEQ ID NO:956) plus one random nucleotide (N) plus the 933 6-mers (SEQ ID NOS:1-933).
Second strand synthesis was carried out using the following primer pool:
5′ CCACTCCATTTGTTCGTGTGNNNNNN 3′ (SEQ ID NO:957) (comprising random 9-mers plus a second PCR primer binding site.)
PCR amplification of the double-stranded cDNA was then carried out using the following PCR primers:
Forward PCR Primer: 5′ CCGAACTACCCACTTGCATT 3′ (SEQ ID NO:958) (binds to first PCR primer binding site)
Reverse PCR Primer: 5′ CCACTCCATTTGTTCGTGTG 3′ (SEQ ID NO:959) (binds to second PCR primer binding site)
Detailed Methods
Test Samples: Test samples 1-8 were carried out using Primer sets 1-8 shown above in TABLE 35 and were tested in parallel as described for primer set #1 below.
Control samples were included as follows:
1. A first strand reverse transcriptase reaction into second strand Klenow, but no N9 primer, followed by PCR amplification.
2. A first strand reverse transcriptase reaction, no second strand Klenow, followed by PCR amplification.
First Strand Reverse Transcription:
First strand reverse transcription was carried out as follows:
Combine:
Mixed and incubated at 65° C. for 5 minutes, snap chilled on ice.
Added 10 μl of RT cocktail (prepared on ice) containing:
The sample was mixed and transferred to a 40° C. pre-warmed thermal cycler (to provide a “hot start”), and the sample was then incubated at 40° C. for two hours, 70° C. for 10 minutes and chilled to 4° C.
1 μl of RNAse H (1-4 units/μl) was then added and the sample was incubated at 37° C. for 20 minutes, then heated to 95° C. for 5 minutes and snap chilled at 4° C.
Second Strand Synthesis
A second strand synthesis cocktail was prepared as follows:
80 μl of the second strand synthesis cocktail was added to the 20 μl first strand template reaction mixture, mixed and incubated at 37° C. for 30 minutes.
cDNA Purification
The resulting double stranded cDNA was purified using Spin Cartridges obtained from Invitrogen and buffers supplied in the kit according to the manufacturer's directions. A total volume of 30 μl was eluted from the column, of which 20 μl was used for follow-on PCR.
PCR Amplification
The following mixture was added to the 20 μl of purified cDNA template:
PCR Amplification Conditions:
25 total PCR cycles:
10 cycles of:
15 cycles of:
Results: The results were analyzed in terms of (1) measuring the level of amplification of selected reporter genes by qPCR, (2) measuring amplified DNA “aDNA” yield, and (3) evaluation of an aliquot of the aDNA on an agarose gel to confirm that the population of species in the cDNA were equally represented.
The data in TABLE 36 shows the raw abundance values (adjusted for dilution) for reporter genes GAPDH, GUSB, hPO and Actin following qPCR of the double-stranded cDNA and aDNA products for samples 1-10.
The raw data shown above in Table 36 was then adjusted based on the following amplification factor:
(PCR/cDNA)*(cDNA/average cDNA)
This amplification factor was introduced to weight amplification in favor of samples that had robust cDNA synthesis levels and robust PCR yields (e.g., sample 2), over samples with poor cDNA and PCR yields, but high levels of cDNA to PCR amplification (e.g., sample 7). The abundance values, adjusted based on the amplification factor described above, are shown below in TABLE 37.
As shown above in TABLE 37, PCR yield was generally good, especially with Primer Sets 1 and 2. Amplification was observed for all reporter genes tested. An aliquot of the aDNA from samples 1-8 and controls 1-2 were run on 1.6% agarose gels (not shown) and each test sample showed a smear of amplified material, indicating that the amplified products were representative of mRNA species present in the starting total RNA.
A second set of reactions were run with the same samples using the methods described above, but with the PCR annealing temperature raised to 60° C. with a total of 30 cycles of PCR. qPCR was performed on the resulting aDNA using qPCR for the eight reporter genes shown in TABLE 38 that are known to be highly expressed in Jurkat cells.
As shown in TABLE 39, the overall yield of aDNA obtained using an annealing temperature at 60° C. during PCR amplification is greater than that obtained with 55° C. for seven out of the eight primer sets tested.
As shown in TABLE 40, quantitative PCR (qPCR) analysis of the aDNA from PCR reactions annealed at 55° C. or 60° C. showed that primer pair 1 at a 60° C. annealing temperature provided the best PCR specificity as measured by the ratio of aDNA to cDNA. It was further determined that the specific activity of reporter genes tracked closely with aDNA yields over the range of PCR cycles tested (4 cycles to 24 cycles) (data not shown).
This Example demonstrates that aDNA amplified from double stranded cDNA templates generated using the 933 6-mers (SEQ ID NOS:1-933), as described in EXAMPLE 11, preserved the expression ratios observed in the double-stranded cDNA templates.
Methods:
First strand cDNA synthesis was performed as described in Example 11 using 1 μg of total RNA template from Jurkat or K562 cells (obtained from Ambion, Inc. Austin Tex.) with the NSR7 primer pool. Two Jurkat samples were run in parallel. For second strand synthesis and cDNA purification were also carried out as described in Example 11.
PCR Amplification of cDNA
The cDNA template was diluted 10-fold and 100-fold prior to use in PCR reaction, and either 10 μl purified, or 10 μl diluted cDNA was used.
Add to the 10 μl of cDNA template:
PCR Amplification Conditions:
Perform 30 total PCR cycles:
10 cycles of:
20 cycles of:
Post-PCR clean-up: 100 μl PCR reaction was diluted with 500 μl buffer and purified using Spin Cartridges obtained from Invitrogen and buffers supplied in the kit according to the manufacturer's directions. A total volume of 55 μl was eluted from the column.
Results: The results were analyzed in terms of total aDNA yield, evaluation of aDNA on agarose gels to verify the presence of the expected smear indicating amplification of substantially all species in the transcriptome, and by measuring a panel of reporter genes by qPCR.
Results: As shown in TABLE 41, the yields of aDNA were consistent across the range of template inputs. These results indicate that the PCR reaction was saturated at the lower template input. The agarose gel analysis showed the expected smear indicating good amplification across the transcriptome (not shown).
In order to determine if the aDNA generated from the cDNA preserved the target genes expression levels present in the cDNA, quantitative PCR analysis was conducted with 54 randomly chosen TaqMan reagents, detecting Gene IDS #1-34. As shown in TABLE 42, of the 54 randomly chosen target genes, measurable signal was measured for 34 genes in both NSR7-primed cDNA and aDNA generated therefrom (from 10 μl cDNA template input). The gene expression signal shown below in Table 42 was calculated as power (10 (log 10(½)*Ct+10)).
As shown in TABLE 42, the results of the Jurkat and K562 experiments show a good correlation in expression ratios between the cDNA and the aDNA derived from the cDNA.
This Example demonstrates that aDNA amplified from double stranded cDNA templates generated using the 933 6-mers (SEQ ID NOS:1-933), as described in EXAMPLE 11, preserved the enrichment of target genes relative to rRNA and globin.
Rationale: To determine whether the enrichment of target genes relative to rRNA and globin previously observed from in-vitro transcription products generated from NSR-primer cDNA is preserved in amplified DNA product generated from PCR amplification of the NSR-primed cDNA.
Methods: In order to determine whether the enrichment of target genes relative to rRNA and globin previously observed from in-vitro transcription products generated from NSR-primer cDNA is preserved in amplified DNA product generated from PCR amplification of the NSR-primed cDNA, the amplified aDNA product obtained from cDNA generated using NSR7 primers was compared to the amplified aDNA product obtained from cDNA generated using random 8-mers in the following experiment.
First strand cDNA synthesis was performed on 1 μg of total RNA isolated from whole blood, as described in Example 11 with duplicate samples generated with either (1) the NSR7 primer pool (comprising SEQ ID NOS:1-933 that each had the T7 promoter (SEQ ID NO: 934) covalently attached at the 5′ end); or (2) a random 8-mer (N8) primer pool with the T7 promoter (SEQ ID NO: 934) covalently attached at the 5′ end.
Second strand synthesis and cDNA purification was performed as described in Example 11, with either SEQ ID NO: 947 for both the NSR7 primed samples, and the random 8-mers (N8) primed samples.
PCR Amplification of cDNA
Either 1 μl+9 μl H20 or 10 μl cDNA template was used in the follow-on PCR reaction as follows:
Add to the 10 μl of cDNA template:
PCR Amplification Conditions:
Perform 20 total PCR cycles:
10 cycles of:
10 cycles of:
Post-PCR clean-up: 100 μl PCR reaction was diluted with 500 μl buffer and purified using Spin Cartridges obtained from Invitrogen and buffers supplied in the kit according to the manufacturer's directions. A total volume of 50 μl was eluted from the column.
Results: The data in TABLE 43 shows the ratio of qPCR abundance values for NSR-primed to N8-primed cDNA (adjusted for dilution) for the following reporter genes: A=IGF1R; B=GAPDH, C═STMN1, and D=CDCA7 compared to background gene expression measured for 18S, 28S, HBA1, HBA2 or HBB.
The data shown above in TABLE 43 shows a dramatic enrichment of target genes over background expression in aDNA amplified from NSR7 primed cDNA. The observed enrichment is consistently better with lower template inputs. Overall, it is clear that the three step amplification method (NSR-PCR) described in EXAMPLE 11 provides a significant enrichment of target genes over rRNAs and globins. Therefore, the NSR-PCR method described in EXAMPLE 11 could be used for full transcriptome profiling of total RNA in a biological sample, such as whole blood.
This Example shows that the yield of amplified DNA can be increased in a PCR reaction through the use of an elevated concentration of primers, dNTPs and MgCl2, thereby allowing for the implementation of NSR-PCR in high-throughput applications.
Rationale: An Experiment was carried out to measure total aDNA yields from a PCR reaction using a cDNA template as a function of primer input concentrations at two different dNTP and Mg++ concentrations as follows.
Methods:
Template: cDNA was prepared from Jurkat total RNA using NSR7 primers, as described in Example 13.
PCR Primers:
Forward: SEQ ID NO:949; Reverse: SEQ ID NO:948
Primer Concentrations tested: 200, 400, 600, 800, and 1000 nM
dNTP concentrations tested: 200 μM, 1.5 mM
Mg++ concentrations tested: 500 μM, 4 mM
PCR reactions were prepared as described in Example 13.
PCR Amplification Conditions:
Perform 30 total PCR cycles:
10 cycles of:
and
20 cycles of:
Post-PCR clean-up: 100 μl PCR reaction was diluted with 500 μl buffer and purified using Spin Cartridges obtained from Invitrogen and buffers supplied in the kit according to the manufacturer's directions. A total volume of 50 μl was eluted from the column.
The results shown above in TABLE 44 indicate that the yields of aDNA at 800 nM primer, 500 uM dNTPs and 4 mM Mg++ approach 10 μg per a 100 μl PCR reaction. The aDNA was also analyzed by agarose gel and was confirmed to contain a smear of amplified material, indicating that the amplified products were representative of mRNA species present in the starting total RNA.
In order to further assess the presence of amplified full transcript material versus the amplification of non-specific material, the panel of genes 1-8 shown in TABLE 38 were analyzed by qPCR. Control sample 9 was 18S rRNA and control sample 10 was 28S rRNA?
These data demonstrate that the high yield of aDNA generated using the optimized PCR conditions contain amplified full transcript material, and the increased yield is not due to the presence of non-specific material generated during the amplification reaction.
This Example describes methods that are useful to label the aDNA PCR products for subsequent use in gene expression monitoring applications.
1. Direct Chemical Coupling of Fluorescent Label to the PCR Product
Cy3 and Cy5 direct label kits were obtained from Mirus (Madison, Wis., kit MIR product numbers 3625 and 3725).
10 μg of PCR product (aDNA), obtained as described in Example 11, was incubated with labeling reagent as described by the manufacturer. The labeling reagents covalently attached Cy3 or Cy5 to the nucleic acid sample, which can then be used in almost any molecular biology application, such as gene expression monitoring. The labeled aDNA was then purified and its fluorescence was measured relative to the starting label.
Results: Four aDNA samples were labeled as described above, and fluorescence was measured. A range of 0.9 to 1.5% of retained label was observed across the four labeled aDNA samples (otherwise referred to as a labeling efficiency of 0.9 to 1.5%). These results fall within the 1% to 3% labeling efficiency typically observed for aaUTP labeled, in vitro translated, amplified RNA.
2. Incorporation of aminoallyl modified dUTP (aadUTP) during PCR with an aDNA template using one primer (forward or reverse) to yield aa-labeled, single-stranded aDNA.
Methods: 1 μg of the aDNA PCR product, generated using the NSR7 primer pool as described in Example 11, is added to a PCR reaction mix as follows:
PCR Reaction: 5 to 20 cycles of PCR (94° C. 30 seconds, 60° C. 30 seconds, 72° C. 30 seconds), during which time only one strand of the double-stranded PCR template is synthesized. Each cycle of PCR is expected to produce one copy of the aa-labeled, single-stranded aDNA. This PCR product is then purified and a Cy3 or Cy5 label is incorporated by standard chemical coupling.
3. Incorporation of aminoallyl modified dUTP (aadUTP) during PCR with an aDNA template using forward and reverse primers to yield aa-labeled, double-stranded aDNA.
Methods: 1 μg of the aDNA PCR product, generated using the NSR7 primer pool as described in Example 11, is added to a PCR reaction mix as follows:
PCR Reaction: 5 to 20 cycles of PCR (94° C. 30 seconds, 60° C. 30 seconds, 72° C. 30 seconds), during which time both strands of the double-stranded PCR template are synthesized. The double-stranded, aa-labeled aDNA PCR product is then purified and a Cy3 or Cy5 label is incorporated by standard chemical coupling.
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the scope of the invention.
This application claims the benefit of U.S. Provisional Application No. 60/790,458, filed Apr. 7, 2006, and also claims the benefit of U.S. Provisional Application No. 60/731,529, filed Oct. 27, 2005.
| Number | Date | Country | |
|---|---|---|---|
| 60731529 | Oct 2005 | US | |
| 60790458 | Apr 2006 | US |
