Patent Application
20060094025
Recently, a class of small non-coding RNAs, termed microRNAs (miRNAs), has been identified that function in post-transcriptional regulation of gene expression in plants and amimals (Carrington and Ambrose, Science 301:336 (2003)). Originally identified in C. elegans, miRNAs act by basepairing to complementary sites in the 3′ untranslated region (UTR) or coding sequences of their target mRNAs and repressing their translation (Wang et al., Nucleic. Acids Res. 32:1688 (2004)).
While mature miRNAs are only ˜22 nucleotides (nt) in length, they originate from hairpin regions of ˜70mer precursor (pre-miRNA) sequences through the action of Dicer complex (Lee et al., EMBO J. 21:4663 (2002)). The mature miRNA is then incorporated into the miRNP, the ribonucleoprotein complex that mediates miRNA's effects on gene regulation (Mourelatos et al., Genes Dev. 16:720 (2002)).
Bioinformatics studies predict that there are ˜100 miRNAs encoded in the worm and fly genomes, and ˜250 miRNAs encoded in the vertebrate genomes. (Lai et al., Genome Biol. 4:R42 (2003); Lim et al., Genes Dev. 17:991 (2003); Lim et al., Science 299:1540 (2003)) This accounts for ˜0.5-1% of the number of predicted protein-coding genes for each genome, underlining the importance of miRNAs as a class of regulatory gene products (Brennecke and Cohen, Genome Biol. 4:228 (2003)).
miRNAs have been implicated in a variety of biological processes, including flower and leaf development in plants, larval development in worms, apoptosis and fat metabolism in flies, and hematopoietic differentiation and neuronal development in mammals (Bartel, Cell 116:281 (2004)). In addition, many miRNA genes map to chromosomal regions in humans associated with cancer (e.g., fragile sites, breakpoints, regions of loss of heterozygosity, regions of amplification) (Calin et al., Proc. Natl. Acad. Sci. USA 101:2999 (2004)). Various miRNAs have also been shown to interact with the fragile X mental retardation protein (FMRP) in vivo (Jin et al., Nat. Neurosci. 7:113 (2004)), suggesting a role for these tiny RNAs in human health and disease.
Because different cell types and disease states are associated with expression of certain miRNAs, it is important to obtain both temporal and spatial expression profiles for miRNAs. Northern hybridization has been used to determine the expression levels of miRNAs (see, e.g., Sempere et al., Genome Biol. 5:R13 (2004); Aravin et al., Dev. Cell 5:337 (2003); Grad et al., Mol. Cell 11;1253 (2003); Lim et al., Genes & Dev. 17:991 (2003)), but this method is too labor intensive for high-throughput analyses. PCR-based methods have been used to monitor the expression of miRNAs, but these methods either require the use of costly gene-specific primers (see, e.g., Schmittgen et al., Nucleic Acids Res. 32:e43 (2004)) or inefficient blunt-end ligations to attach primer-binding linkers to the miRNA molecules (see, e.g., Miska et al., Genome Biol. 5:R68 (2004); Grad et al., Mol. Cell 11;1253 (2003); Lim et al., Genes & Dev. 17:991 (2003)). In addition, PCR can introduce significant biases into the population of amplified target miRNA molecules.
High-throughput microarrays have recently been developed to identify expression patterns for miRNAs in a variety of tissue and cell types (see, e.g., Babak et al., RNA 10:1813 (2004); Calin et al., Proc. Natl. Acad. Sci. USA 101:11755 (2004); Liu et al., Proc. Natl. Acad. Sci. USA 101:9740 (2004); Miska et al., Genome Biol. 5:R68 (2004); Sioud and RØsok, BioTechniques 37:574 (2004); Krichevsky et al., RNA 9:1274 (2003)). The use of microarrays has several advantages for detection of miRNA expression, including the ability to determine expression of multiple genes in the same sample at a single time point, a need for only small amounts of RNA, and the potential to simultaneously identify the expression of both precursor and mature miRNA molecules.
However, since mature miRNAs are only ˜22 nt in length and present in very limited quantities in any given tissue, these small RNAs present challenges for microarray labeling and detection (Sioud and RØsok, BioTechniques 37:574 (2004)). For example, covalent attachment of fluorophores can be used to directly label miRNA molecules for use in microarray analyses (see, e.g., Babak et al., RNA 10:1813 (2004); MICROMAX ASAP miRNA Chemical Labeling Kit, Perkin Elmer, Shelton, Conn.; Label IT® μArray Labeling Kit, Mirus Bio Corp., Madison, Wis.), but this method lacks the sensitivity to detect rare target miRNA molecules. Direct labeling can also result in intermolecular quenching of the randomly incorporated fluorophores, resulting in further decreased sensitivity. Random primed-reverse transcription of miRNA molecules has been used to produce labeled cDNA molecules for use in microarray analyses (see, e.g., Sioud and RØsok, BioTechniques 37:574 (2004); Liu et al., Proc. Natl. Acad. Sci. USA 101:9740 (2004)), but this method does not yield an accurate representation of the original full-length miRNA population. As a result, there is an immediate need for sensitive and efficient methods for labeling and detection of miRNA molecules for use in microarray analyses.
Applicants have invented methods for the labeling of target miRNA molecules and cDNA molecules complementary to target miRNA molecules for use in microarray analyses, wherein a capture sequence complementary to a capture reagent sequence is attached either directly to the 3′ end of the miRNA molecules or to the 5′ end of cDNA molecules complementary to the miRNA molecules. A capture reagent containing a label and the capture reagent sequence is then attached to the capture sequence, creating labeled nucleic acid molecules for use in miRNA microarray analyses. Applicants have discovered that quenching can be reduced and signal intensity enhanced without the need for PCR through the use of a capture sequence and a labeled capture reagent, resulting in improved methods and reagents for miRNA microarray analyses.
One aspect of this invention is directed to a method for producing a labeled target miRNA molecule comprising:
Another aspect of this invention is directed to a method for producing a labeled cDNA molecule complementary to a target miRNA molecule comprising:
In some embodiments, the capture reagent is attached to the capture sequence following hybridization of the target nucleic acid to a microarray containing at least one sense or antisense miRNA probe. In other embodiments, the capture reagent is attached to the capture sequence prior to hybridization.
Applicants have also invented methods for the detection of miRNA probes on a microarray using target miRNA molecules and cDNA molecules complementary to target miRNA molecules containing a capture sequence complementary to a capture reagent sequence. Prior to or following hybridization of the capture sequence-tagged nucleic acid molecules to the microarray, a capture reagent containing a label and the capture reagent sequence is attached to the capture sequence, allowing for the detection of miRNA sense and antisense probes on the microarray.
One aspect of this invention is directed to a method for the detection of a miRNA antisense probe on a microarray comprising:
Another aspect of this invention is directed to a method for the detection of a miRNA sense probe on a microarray comprising:
In some embodiments, the capture reagent is attached to the capture sequence following hybridization of the target nucleic acid to the miRNA probes on the microarray. In other embodiments, the capture reagent is attached to the capture sequence prior to hybridization.
Applicants have also invented kits for the production of labeled target miRNA molecules and cDNA molecules complementary to target miRNA molecules for use in microarray analyses, wherein a capture sequence complementary to a capture reagent sequence is attached either directly to the 3′ end of the miRNA molecules or to the 5′ end of cDNA molecules complementary to the miRNA molecules.
One aspect of this invention is directed to a kit for the production labeled target miRNA molecules for use in microarray analyses comprising: a partially double stranded nucleic acid sequence having a sense strand and antisense strand, wherein the sense strand comprises a capture sequence and the antisense strand comprises a single stranded 3′ overhang comprising a sequence complementary to an oligonucleotide tail; and instructional materials for producing a labeled target miRNA molecule using the partially double stranded nucleic acid sequence.
In some embodiments, the kit comprises at least one enzyme for attaching an oligonucleotide tail onto the 3′ end of a single stranded target miRNA molecule, wherein the oligonucleotide tail is complementary to the single stranded 3′ overhang sequence of the partially double stranded nucleic acid sequence; and at least one enzyme for attaching the 5′ end of the sense strand of the partially double stranded nucleic acid sequence to the 3′ end of the single stranded target miRNA molecules. In further embodiments, the kit comprises a capture reagent comprising a label capable of emitting a detectable signal and a nucleic acid sequence complementary to the capture sequence. In still further embodiments, the kit comprises components, reagents and instructional materials for use of the labeled target miRNA molecules in a microarray assay.
Another aspect of this invention is directed to a kit for the production of labeled cDNA molecules complementary to target miRNA molecules for use in microarray analyses comprising: a single stranded primer comprising a capture sequence at its 5′ end and a sequence complementary to an oligonucleotide tail at its 3′ end; and instructional materials for producing the labeled cDNA molecules complementary to target miRNA molecules using the single stranded primer.
In some embodiments, the kit further comprises at least one enzyme for attaching an oligonucleotide tail onto the 3′ end of a single stranded cDNA molecule complementary to a target miRNA molecule, wherein the oligonucleotide tail is complementary to the 3′ end of the single stranded primer. In some embodiments, the kit comprises at least one reverse transcriptase for extending the single stranded primer from its 3′ end to produce a single stranded cDNA molecule complementary to a target miRNA molecule comprising a capture sequence at its 5′ end. In further embodiments, the kit comprises a capture reagent comprising a label capable of emitting a detectable signal and a nucleic acid sequence complementary to the capture sequence. In still further embodiments, the kit comprises components, reagents and instructional materials for use of the labeled cDNA molecules complementary to target miRNA molecules in a microarray assay.
a-g together depict labeling of a target miRNA molecule or a cDNA molecule complementary to a target miRNA and the detection of sense and antisense miRNA probes according to the methods of the present invention.
The present invention relates to nucleic acid molecules, methods and kits for use in miRNA microarray analyses. The terms “RNA molecule”, “miRNA molecule” “mRNA molecule”, “DNA molecule”, “cDNA molecule”, and “nucleic acid molecule” are each intended to cover a single molecule, a plurality of molecules of a single species, and a plurality of molecules of different species. The term “miRNA molecule” is also intended to cover both mature and pre-miRNA molecules. Consistent with microarray terminology, “target miRNA” refers to a miRNA or complementary cDNA sequence to be labeled, while “miRNA probe” refers to an unlabeled sense or antisense miRNA sequence attached directly to a microarray support. The term “capture sequence” refers to any non-native nucleotide sequence capable of binding to a “capture reagent sequence”, while the term “capture reagent” refers to a reagent containing a detectable molecule or molecules and a capture reagent sequence or sequences complementary to the capture sequence.
The methods of the present invention comprise attaching a capture sequence onto the 3′ end of at least one miRNA molecule or 5′ end of at least one cDNA molecule complementary to at least one miRNA molecule; and attaching to the capture sequence a capture reagent comprising a label capable of emitting a detectable signal and a nucleotide sequence complementary to the capture sequence. The resulting labeled miRNA and cDNA molecules are then used to detect sense or antisense miRNA probes attached to microarrays, allowing miRNA expression profiles to be obtained. By using appropriately labeled target molecules and appropriately designed probes, the both mature and pre-miRNA expression profiles can be determined.
The methods of the present invention are distinct over currently available technologies that directly label target miRNA molecules by covalent attachment of fluorophores or that random prime and reverse transcribe target miRNA molecules to produce labeled cDNA molecules, both of which lack the sensitivity necessary for detecting rare target miRNA molecules following hybridization to miRNA probes. The methods of the present invention are also distinct over PCR-based labeling technologies, which can introduce amplification bias into the population of labeled target molecules.
The methods of the present invention utilize routine techniques in the field of molecular biology. Basic texts disclosing general molecular biology methods include Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001) and Ausubel et al., Current Protocols in Molecular Biology (1994).
The methods of the present invention utilize sources of RNA molecules. Preferably, the sources are enriched for miRNA molecules. Numerous methods and commercial kits are available for the enrichment of miRNA molecules from total RNA. Examples include the miRvana™ miRNA Isolation Kit (Ambion, Austin, Tex.), purification on denaturing PAGE gels (see, e.g., Miska et al., Genome Biol. 5:R68 (2004)), centrifugation with appropriately sized molecular weight cutoff filters (e.g., Microcon® YM filter devices, Millipore, Billerica, Mass.), and sodium acetate/ethanol precipitation (see, e.g., Wang et al., Nucleic Acids Res. 32:1688 (2004)). The miRNA may be obtained from any tissue or cell source that contains miRNA, including virion, plant, and animal sources found in any biological or environmental sample. Preferably, the source is animal tissue, more preferably mammalian tissue, most preferably human tissue.
With reference to
To produce labeled target miRNA molecules, a partially double stranded deoxynucleic acid sequence containing a sense strand capture sequence is attached to the 3′ oligonucleotide tail by ligation (see
Alternatively, to produce labeled cDNA molecules complementary to target miRNA molecules, a single stranded primer comprising a capture sequence at its 5′ end and a sequence complementary to the oligonucleotide tail at its 3′ end is annealed to the 3′ oligonucleotide tail on the miRNA molecules (see
Following the cDNA synthesis, the RNA is generally degraded prior to purification of the first strand cDNA molecules (see
The capture sequence-tagged miRNA or cDNA molecules are then contacted with a microarray containing either sense or antisense miRNA probes (see
As used herein, “microarray” is intended to include any solid support containing nucleic acid probes, including slides, chips, membranes, beads, and microtiter plates. Methods for attaching miRNA probes to solid supports are well known to those of skill in the art (see, e.g., Babak et al., RNA 10:1813 (2004); Calin et al., Proc. Natl. Acad. Sci. USA 101:11755 (2004); Liu et al., Proc. Natl. Acad. Sci. USA 101:9740 (2004); Miska et al., Genome Biol. 5:R68 (2004); Sioud and RØsok, BioTechniques 37:574 (2004); Krichevsky et al., RNA 9:1274 (2003)). Alternatively, miRNA microarrays can be obtained commercially from, e.g., The Neuroscience Gene Expression Laboratory, W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, N.J.
Sense and antisense miRNA probes can be designed using known miRNA and pre-miRNA sequences publicly available from, e.g., The miRNA Registry, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK (Griffiths-Jones, Nucleic Acids. Res. 32:D109 (2004). Novel miRNA sequences can also be used to design miRNA probes and can be identified using computational methods (see, e.g., Ambros et al., Curr. Biol. 13:807 (2003); Grad et al., Mol. Cell 11;1253 (2003); Lai et al., Genome Biol. 4:R42 (2003); Lim et al., Genes & Dev. 17:991 (2003); Lim et al., Science 299:1540 (2003)) or miRNA cloning strategies (see, e.g., Wang et al., Nucleic Acids Res. 32:1688 (2004); Lagos-Quintana et al., Science 294:853 (2001); Lau et al., Science 294:858 (2001); Lee et al., Science 294:862 (2001)) well known to those skilled in the art.
Following hybridization of either the capture-sequence tagged miRNA or cDNA molecules to the sense or antisense miRNA probes on the microarray, a capture reagent containing a label capable of emitting a detectable and at least one capture reagent sequence complementary to the capture sequence is attached to the hybridized capture sequence-tagged nucleic acid molecules (see
The label of the capture reagent comprises one or more molecules capable of emitting a detectable signal. By using a capture reagent, the signal-producing molecule or molecules can be positioned such that quenching is reduced or eliminated. Furthermore, the signal in the capture reagent can be amplified or enhanced without bias-introducing amplification of the target nucleic acid molecules themselves. Preferably, the capture reagent is a 3DNA™ Dendrimer Capture Reagent (Genisphere Inc., Hatfield, Pa.). Dendrimers are highly branched nucleic acid molecules that contain two types of single stranded hybridization “arms” on their surface for the attachment of a label and a capture sequence. Because a single dendrimer may have hundreds of arms of each type, the signal obtained upon hybridization is greatly enhanced. Signal enhancement using dendritic reagents is described in Nilsen et al., J. Theor. Biol. 187:273 (1997); Stears et al., Physiol. Genomics, 3:93 (2000); U.S. Pat. Nos. 5,175,270, 5,484,904, 5,487,973, 6,072,043, 6,110,687, and 6,117,631; and U.S. Patent Publication No. 2002/0051981.
The signal producing molecule can be any molecule capable of emitting or producing a detectable signal. Such molecules include those that directly emit or produce a detectable signal, such as radioactive molecules, fluorescent molecules, and chemiluminescent molecules, as well as enzymes used in colorimetric assays, such as horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Such molecules also include those that do not directly produce a detectable signal but which bind in systems that do, such as biotin/streptavidin and antigen/antibody. Preferably, the signal-producing molecule is one that directly emits or produces a detectable signal, more preferably a fluorophore, most preferably a Cy3 or Cy5 dye or other suitable dye, such as Alexa Fluor® 555 or 647 dyes (Molecular Probes, Inc., Eugene, Oreg).
Although
Following attachment of the capture reagent to the miRNA probes on the microarray, the microarray is washed to remove unhybridized labeled miRNA or cDNA molecules and the resulting hybridization pattern visualized by detection of the signal from the hybridized labeled nucleic acid molecules (see
The methods and reagents of the present invention can be conveniently packaged in kit form. Such kits can be used in various research and diagnostic applications. For example, methods and kits of the present invention can be used to facilitate a comparative analysis of expression of one or more miRNAs in different cells or tissues, different subpopulations of the same cells or tissues, different physiological states of the same cells or tissue, different developmental stages of the same cells or tissue, or different cell populations of the same tissue. Such analyses can reveal statistically significant differences in the levels of miRNA expression, which, depending on the cells or tissues analyzed, can then be used to facilitate diagnosis of various disease states, prognosis of disease progression, and identification of targets for disease treatment.
A wide variety of kits may be prepared according to the present invention. For example, a kit for the production of labeled target miRNA molecules may include a partially double stranded nucleic acid sequence having a sense strand and antisense strand, wherein the sense strand comprises a capture sequence and the antisense strand comprises a single stranded 3′ overhang comprising a sequence complementary to an oligonucleotide tail; and instructional materials for producing labeled target miRNA molecules using the partially double stranded nucleic acid sequence.
A kit for the production of labeled cDNA molecules complementary to target miRNA molecules may include a single stranded primer comprising a capture sequence at its 5′ end and a sequence complementary to the oligonucleotide tail at its 3′ end; and instructional materials for producing labeled cDNA molecules complementary to target miRNA molecules using the single stranded primer.
While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The kits may also include one or more of the following components or reagents for production of the labeled miRNA and cDNA molecules of the present invention: an RNase inhibitor; an enzyme for attaching an oligonucleotide tail onto single stranded miRNA molecules (e.g., poly(A) polymerase); a reverse transcriptase; an enzyme for attaching the partially double stranded nucleic acid sequence to the oligonucleotide tail (e.g., T4 DNA ligase); and a capture reagent comprising a label capable of producing or emitting a detectable signal and a nucleic acid sequence complementary to the capture sequence. The kits may further include components and reagents and instructional materials for use of the labeled miRNA and cDNA molecules in microarray assays, including hybridization and wash solutions, incubation containers, cover slips, and various signal-detecting, signal-producing, signal-enhancing, and signal-preserving reagents. Additionally, the kits may include buffers, nucleotides, salts, RNase-free water, containers, vials, reaction tubes, and the like compatible with the production and use of the labeled nucleic acid molecules of the present invention. The components and reagents may be provided in numbered containers with suitable storage media.
Specific embodiments according to the methods of the present invention will now be described in the following examples. The examples are illustrative only, and are not intended to limit the remainder of the disclosure in any way.
Tailing of miRNA
Briefly, up to 150 ng of rat brain miRNA purified using the miRvana™ miRNA Isolation Kit (Ambion) was adjusted to 15.5 μl with nuclease-free water and mixed with 5 μl 5× Reaction Buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2), 5 μl 25 mM MnCl2, 1 μl 10 mM ATP, and 1 μl (5 U) poly(A) polymerase. The mixture was briefly mixed, centrifuged and incubated in a 37° C. heat block for 15 min.
Ligation of miRNA
The tailed miRNA mixture was briefly centrifuged and mixed with 5 μl Cy3 or Cy5 Ligation Mix (Genisphere) in 5.5× Ligation Buffer (Roche Applied Science, Indianapolis, Ind.) and 2 μl T4 DNA ligase. The Cy3 Ligation Mix contained a sense strand oligonucleotide (175 ng/μl) having a sequence of 5′-PO4-TTC TCG TGT TCC GTT TGT ACT CTA AGG TGG A-3′ (SEQ ID NO:1) and an antisense strand oligonucleotide (271 ng/μl) having a sequence of 5′-ACA CGA GAA TTT TTT TTT T-3′ (SEQ ID NO:2). The Cy5 Ligation Mix contained a sense strand oligonucleotide (175 ng/μl) having a sequence of 5′-PO4-ATT GCC TTG TAA GCG ATG TGA TTC TAT TGG A-3′ (SEQ ID NO:3) and an antisense strand oligonucleotide (271 ng/μl) having a sequence of 5′-CAA GGC AAT TTT TTT TTT T-31 (SEQ ID NO:4). Each set of sense and antisense oligonucleotides was designed to form a partially double stranded nucleic acid molecule having a sense strand containing a capture sequence at its 3′ end and an antisense strand containing 3′ overhang sequence complementary to the poly(A) tail on the target miRNA molecules. The mixture was briefly mixed, centrifuged and incubated at room temperature for 30 min. The reaction was stopped by addition of 3.5 μl 0.5 M EDTA. The volume was adjusted to 100 μl by addition of 64.5 μl 1× TE buffer, pH 8.0, and the mixture briefly mixed and centrifuged. The tagged miRNA mixture was purified using the Qiagen MinElute™ PCR Purification Kit according to the manufacturer's protocol and eluted with 10 μl EB buffer.
Tagged miRNA Microarray Hybridization
Prior to preparing microarray hybridization mixtures, the 2× SDS-based Hybridization Buffer (2×SSC, 4× Denhardt's Solution, 1% SDS, 0.5 M sodium phosphate, 2 mM EDTA, pH 8.0) and 2× Enhanced Hybridization Buffer (ExpressHyb™ buffer (BD Biosciences Clontech, Palo Alto, Calif.) diluted to 75% with nuclease-free water) were thawed and resuspended. Dual color microarray hybridization mixtures using these buffers were prepared according to the tables below:
For single color assays, only one dye-tagged miRNA population is included in the chosen hybridization mixture, with the remaining volume made up with nuclease free water.
The chosen hybridization mixture was gently mixed, briefly centrifuged, and heated first at 75-80° C. for 10 min and then at hybridization temperature (50-54° C.) prior to microarray loading. A microarray was prepared by spotting 22-50 mer antisense miRNA probes representing mature rat miRNA sequences (Compugen, Jamesburg, N.J.) onto a poly-L-lysine-coated slide in 1×SSC buffer. The hybridization mixture was gently mixed, briefly centrifuged, and applied to the microarray prewarmed at 50-54° C., taking care to leave any precipitate at the bottom of hybridization mixture tube. After applying a cover slip, the microarray was incubated overnight at 50-54° C. in a dark humidified glass Coplin jar.
The coverslip was removed by washing the microarray in 2×SSC, 0.2% SDS wash buffer prewarmed to 42° C. The microarray was sequentially washed in prewarmed 2×SSC, 0.2% SDS wash buffer for 15 min, 2×SSC for 10-15 min at room temperature, and 0.2×SSC for 10-15 min at room temperature. The microarray was transferred to a dry 50 mL centrifuge tube, orienting the slide so that any adhesive bar code or label was down in the tube. The tube containing the microarray was immediately centrifuged without the tube cap at 800-1000 rpm to dry the microarray. The microarray was removed from the tube, taking care not to touch the microarray surface.
Capture Reagent Hybridization and Detection
Prior to preparing the capture reagent hybridization mixture, the 3DNA™ Dendrimer Capture Reagent (20 ng/μl) (Genisphere) was thawed and resuspended by vigorous vortexing and heating according to manufacturer's instructions. Each 3DNA Dendrimer Capture Reagent contains numerous conjugated Cy3 or Cy5 fluorophores and oligonucleotides having sequences complementary to the Cy3 (5′-TCC ACC TTA GAG TAC AAA CGG AAC ACG AGA ATT TTT CG-3′; SEQ ID NO: 5) or Cy5 capture sequence (5′-TCC AAT AGA ATC ACA TCG CTT ACA AGG CAA TTT TTT Cg-3′; SEQ ID NO:6). A dual color capture reagent hybridization mixture was prepared according to the table below:
For single color assays, only the single corresponding Capture Reagent is included in the hybridization mixture, with the remaining volume made up with nuclease free water.
The 3DNA™ hybridization mixture was gently mixed, briefly centrifuged, and heated first at 75-80° C. for 10 min and then at 60° C. prior to microarray loading. The microarray was also prewarmed at 60° C. The 3DNA™ hybridization mixture was gently mixed, briefly centrifuged, and applied to the pre-warmed microarray, taking care to leave any precipitate at the bottom of hybridization mixture tube. After applying a cover slip, the microarray was incubated for 3-4 hrs at 60° C. in a dark humidified glass Coplin jar.
The coverslip was removed by washing the microarray in 2×SSC, 0.2% SDS wash buffer prewarmed to 55-60° C. The microarray was sequentially washed in prewarmed 2×SSC, 0.2% SDS wash buffer for 15 min, 2×SSC for 10-15 min at room temperature, and 0.2×SSC for 10-15 min at room temperature. The microarray was transferred to a dry 50 mL centrifuge tube, orienting the slide so that any adhesive bar code or label was down in the tube. The tube containing the microarray was immediately centrifuged without the tube cap at 800-1000 rpm to dry the microarray. The microarray was removed from the tube, taking care not to touch the microarray surface. DyeSaver™2 Anti-Fade Coating (Genisphere) was applied to the microarray to preserve the fluorescent signal and the array scanned using a GenePix™ (Axon Instruments, Union City, Calif.) 4000B microarray scanner with GenePix® Pro 3.0 software, thereby producing an expression profile of the miRNA sequences in the original sample.
Tailing of miRNA
Briefly, up to 150 ng of rat brain miRNA purified using the miRvana™ miRNA Isolation Kit (Ambion) was adjusted to 15.5 μl with nuclease-free water and mixed with 5 μl 5× Reaction Buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2), 5 μl 25 mM MnCl2, 1 μl 10 mM ATP, and 1 μl (5 U) poly(A) polymerase. The mixture was briefly mixed, centrifuged and incubated in a 37° C. heat block for 15 min.
Reverse Transcription of Tailed miRNA
The tailed miRNA mixture was briefly centrifuged and mixed on ice with 2 μl 30 pmoles/μl Cy3 (5′-TTC TCG TGT TCC GTT TGT ACT CTA AGG TGG ATT TTT TTT TTT TTT TTT-3′; SEQ ID NO:7) or Cy5 (5′-ATT GCC TTG TAA GCG ATG TGA TTC TAT TGG ATT TTT TTT TTT TTT TTT-3′; SEQ ID NO:8) reverse transcription (RT) primer (Genisphere). These primers contain a capture sequence at their 5′ ends and a sequence of deoxythymidines complementary to the polyA tail at their 3′ end. The mixture was briefly mixed, centrifuged, incubated at 65° C. for 10 min, and immediately transferred to ice for 2 min. The following reagents were added on ice for a final volume of 50 μl:
10 μl 5× First Strand Buffer (Invitrogen, Carlsbad, Calif.)
5 μl 0.1 M DTT
2.5 μl 10 mM dNTP mix
1 μl Superase-In™ RNase Inhibitor (Ambion)
2 μl (200 units) Superscript™ II reverse transcriptase (Invitrogen)
2.5 μl Nuclease Free Water
The mixture was gently mixed and incubated at 42° C. for 1 hr. The reaction was stopped and the RNA degraded by addition of 8.75 μl 0.5 M NaOH/50 mM EDTA and incubating at 65° C. for 15 min. The reaction was neutralized with 12.5 μl 1 M Tris, pH 8. For dual color assays, the two tagged-cDNA populations were combined into a single tube and briefly mixed and centrifuged. The tagged cDNA was concentrated to 20 μl using a Microcon® YM-10 Centrifugal Filter Device (Millipore) according manufacturer's instruction.
Tagged cDNA Microarray Hybridization
Prior to preparing microarray hybridization mixtures, the 2× SDS-based Hybridization Buffer and 2× Enhanced Hybridization Buffer were thawed and resuspended as described above. Microarray hybridization mixtures using these buffers were prepared according to the tables below:
For single color assays, only one dye-tagged cDNA population is included in the chosen hybridization mixture.
The chosen hybridization mixture was gently mixed, briefly centrifuged, and heated first at 75-80° C. for 10 min and then at hybridization temperature (45-50° C.) prior to microarray loading. A microarray was prepared by spotting 22-50 mer sense miRNA probes representing mature rat miRNA sequences (Compugen) onto a poly-L-lysine-coated slide in 1×SSC buffer. The hybridization mixture was gently mixed, briefly centrifuged, and applied to the microarray prewarmed at 45-50° C., taking care to leave any precipitate at the bottom of hybridization mixture tube. After applying a cover slip, the microarray was incubated overnight at 45-50° C. in a dark humidified glass Coplin jar.
The coverslip was removed by washing the microarray in 2×SSC, 0.2% SDS wash buffer prewarmed to 42° C. The microarray was sequentially washed in prewarmed 2×SSC, 0.2% SDS wash buffer for 15 min, 2×SSC for 10-15 min at room temperature, and 0.2×SSC for 10-15 min at room temperature. The microarray was transferred to a dry 50 mL centrifuge tube, orienting the slide so that any adhesive bar code or label was down in the tube. The tube containing the microarray was immediately centrifuged without the tube cap at 800-1000 rpm to dry the microarray. The microarray was removed from the tube, taking care not to touch the microarray surface.
Capture Reagent Hybridization and Detection
Prior to preparing the capture reagent hybridization mixture, the 3DNA™ Dendrimer Capture Reagent (Genisphere) was thawed and resuspended as described above. A capture reagent hybridization mixture was prepared according to the table below:
For single color assays, only the single corresponding Capture Reagent is included in the hybridization mixture, with the remaining volume made up with nuclease free water.
The 3DNA™ hybridization mixture was gently mixed, briefly centrifuged, and heated first at 75-80° C. for 10 min and then at 60° C. prior to microarray loading. The microarray was also prewarmed at 60° C. The 3DNA™ hybridization mixture was gently mixed, briefly centrifuged, and applied to the pre-warmed microarray, taking care to leave any precipitate at the bottom of hybridization mixture tube. After applying a cover slip, the microarray was incubated for 3-4 hrs at 60° C. in a dark humidified glass Coplin jar.
The coverslip was removed by washing the microarray in 2×SSC, 0.2% SDS wash buffer prewarmed to 55-60° C. The microarray was sequentially washed in prewarmed 2×SSC, 0.2% SDS wash buffer for 15 min, 2×SSC for 10-15 min at room temperature, and 0.2×SSC for 10-15 min at room temperature. The microarray was transferred to a dry 50 mL centrifuge tube, orienting the slide so that any adhesive bar code or label was down in the tube. The tube containing the microarray was immediately centrifuged without the tube cap at 800-1000 rpm to dry the microarray. The microarray was removed from the tube, taking care not to touch the microarray surface. DyeSaver™2 Anti-Fade Coating (Genisphere) was applied to the microarray to preserve the fluorescent signal and the array scanned using a GenePix® (Axon Instruments, Union City, Calif.) 4000B microarray scanner with GenePix® Pro 3.0 software, thereby producing an expression profile of the miRNA sequences in the original sample.
A kit for the production and microarray hybridization of labeled target miRNA molecules was assembled with the following components:
Cy3 and Cy5 3DNA™ Capture Reagent (20 ng/μl) (Genisphere);
5× Reaction Buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2);
MnCl2 (25 mM);
ATP Mix (10 mM);
Poly(A) Polymerase (5 U/μl);
2× SDS-Based Hybridization Buffer (2×SSC, 4× Denhardt's Solution, 1% SDS, 0.5 M sodium phosphate, 2 mM EDTA, pH 8.0);
2× Enhanced Hybridization Buffer (ExpressHyb™ buffer (BD Biosciences Clontech) diluted to 75% with nuclease-free water);
T4 DNA Ligase (1 U/μl);
Cy3 and Cy5 Ligation Mix (75 ng/μl sense oligonucleotide and 271 ng/μl antisense oligonucleotide) (Genisphere); and
Nuclease-Free Water.
The components were placed in numbered vials and placed in a container with a printed instruction manual for the production and microarray hybridization of labeled target miRNA molecules using the kit components.
A kit for the production and microarray hybridization of labeled cDNA molecules complementary to target miRNA molecules was assembled with the following components:
Cy3 and Cy5 3DNA Capture Reagent (20 ng/μl) (Genisphere);
5× Reaction Buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2);
MnCl2 (25 mM);
ATP Mix (10 mM);
Poly(A) Polymerase (5 U/μl);
2× SDS-Based Hybridization Buffer (2×SSC, 4× Denhardt's Solution, 1% SDS, 0.5 M sodium phosphate, 2 mM EDTA, pH 8.0);
2× Enhanced Hybridization Buffer (ExpressHyb™ buffer (BD Biosciences Clontech) diluted to 75% with nuclease-free water);
dNTP Mix (10 mM);
Cy3 and Cy5 RT Primers (30 pmoles/μl) (Genisphere);
Superase-In™ RNase Inhibitor (Ambion); and
Nuclease-Free Water.
The components were placed in numbered vials and placed in a container with a printed instruction manual for the production and microarray hybridization of labeled cDNA molecules complementary to target miRNA molecules using the kit components.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.
