Patent Application
20230416820
The subject matter described herein relates to cellular signaling pathway analysis based on gene expression data. More specifically the subject matter relates to primers, probes and kits which are tailored for establishing the gene expression levels in a sample for the purpose of determining cellular signaling pathway activity. The subject further relates to PCR based methods to determine the expression levels of target genes which can be used for this purpose.
Determining the activity of cellular signaling pathways in a sample is an emerging technology with many applications in diagnosis and prognostics, as well as biotechnological applications. It was found by the inventors that using a mathematical model, the cellular signaling pathway activity can be determined based on the expression levels of target genes of the cellular signaling pathway, as e.g. described in WO2013011479A2, WO2014102668A2, WO2015101635A1, WO2016062891A1, WO2017029215A1, WO2019068585A1, WO2019068562A1, WO2019068543A1 and WO2019120658A1 (each incorporated in its entirety by reference).
For many applications it would be desirable to determine the pathway activities of multiple cellular signaling pathways simultaneously. This means that many expression levels need to be determined in a sample at the same time. Namely, for each pathway preferably at least three target genes as well as at least three reference genes for normalization purposes. This simultaneous determination of expression levels could for example by done using Affymetrix Microarray technology, however this technology has the disadvantage that it is time consuming, expensive and cannot be performed in every lab due to the required equipment and specialized personnel.
The present invention aims, among other, to solve the above problems by the methods and products as defined in the appended claims.
In an aspect, the invention relates to an assembly of primers and probe for determining the activity of the AR cellular signaling pathway, and optionally one or more additional cellular signaling pathway, wherein the assembly of primers and probes comprises three or more sets of primers or probes for determining the expression level of three or more target genes of the AR cellular signaling pathway, wherein said three or more sets of primers and probes are selected from Table 2 of the description,
In an aspect the invention relates to a kit of parts for determining the expression levels for a plurality of genes, the kit comprising primers and probes for the amplification and detection of the expression levels of the plurality of genes, wherein the kit comprises an assembly of primers and probes as defined in the first aspect of the invention, wherein the kit further comprises primers and probes for the amplification and detection of three or more of the reference genes selected from ACTB, ALAS1, B2M, EEF1A1 POLR2A, PUM1, RPLP0, TBP, TPT1 and TUBA1B, preferably wherein said three or more sets of primers and probes are selected from Table 8 of the description, and wherein each primer and/or probe individually has the listed nucleotide sequence identified by the corresponding SEQ ID NO or has a sequence that differs at 1, 2 or 3 positions, wherein said 1, 2 or 3 differences are individually selected from a single base substitution, a single base deletion or a single base addition.
In an aspect the invention relates to the use of the assembly of primers and probes as defined in first aspect or the kit as defined in the second aspect of the invention for determining the AR cellular signaling pathway activity, and optionally the cellular signaling pathway activity of one or more cellular signaling pathways selected from the group consisting of: HH, ER, TGFbeta, PI3K-FOXO, Notch, MAPK-AP1, JAK-STAT1/2 and NFkB.
In an aspect the invention relates to the use of a set of three or more primers and probes to determine the expression levels of three or more target genes of a cellular signaling pathway, wherein the set of primers and probe combinations are as defined in the first aspect of the invention, and
In an aspect the invention relates to a method for designing primers and probes for the detection of the expression levels of target genes of a cellular signaling pathway suitable for determining the activity of the AR cellular signaling pathway and optionally one or more additional cellular signaling pathways, the method comprising:
In a sixth aspect the invention relates to a method of determining the AR cellular signaling pathway activity and optionally one or more additional cellular signaling pathway activity or activities, by simultaneously determining the expression level of six or more genes in a sample, the method comprising simultaneously amplifying six or more gene products using a polymerase chain reaction to generate a plurality of amplification products, followed by the detection of the plurality of amplification products using a plurality of probes,
An alternative to e.g. a microarray based technology would be to use PCR based technology like qPCR, however the problem with such technology is that the used primers and probes often require different conditions for optimal performance, meaning many different reactions need to be performed. The present invention aims to overcome the above problems, among others, by narrowly defining the reaction conditions combined with the selection criteria for the primers and probes, and further by providing sets of primers and probes suitable for the designed reaction conditions which can be used to determine the expression levels of the different target genes which can be used to determining cellular signaling pathway activity. This allows the primers and probes to be used under the same reaction conditions (e.g. in the same multi-well plate or even as a multiplex).
Therefore, in a first aspect the present invention relates to a method for simultaneously determining the expression level of six or more genes in a sample, the method comprising simultaneously amplifying six or more gene products using a polymerase chain reaction to generate a plurality of amplification products, followed by the detection of the plurality of amplification products using a plurality of probes,
One of the advantages of the above method is that it allows the detection of target genes for multiple cellular signaling pathways, and can be performed in a single reaction. This allows the very quick (e.g. within 2-3 hours) detection of expression levels in target genes and subsequent determination of cellular signaling pathway activities. This is very advantageous, for example in critical care, where cellular signaling pathway analysis may be used for quick diagnosis of a patient. In such cases it is essential that the method can be performed fast and without specialized equipment or personnel, as is the case with the disclosed method. The present method allows the detection of the expression levels of target genes in a sample for multiple cellular signaling pathways in a single reaction.
A further advantage of the method as disclosed herein is that it allows reliable determination of expression levels even in samples in which this is typically difficult to do so such as Formalin-Fixed Paraffin-Embedded (FFPE) tissue.
The present method requires a simple qPCR device to run, preferably a thermal cycler with fluorescence readout such as, but not limited to, the Idylla platform. For example a premade plate or container can be used comprising the required reagents and primers and probes, on which the sample is deposited. The PCR protocol does not need to be amended depending on the pathway(s) that are to be analyzed as it is standardized. During amplification cycling, probe intensity can be measured and pathway activity can be determined following conclusion of the protocol, based on the determined expression levels of the target genes. This final step uses a mathematical method to relate the input numerical values representing the expression levels of three or more target genes to a pathway activity. This step is almost instantaneously and can either be performed locally e.g. on a computer or a phone or on a remote server.
Although the method describes target genes for each cellular signaling pathway, it is envisioned that due to the selection criteria for the primers and probes, the method can be applied to alternative target genes for the mentioned cellular signaling pathways that are not listed here. Alternatively, the expression levels of the target genes for cellular signaling pathways not listed herein may be determined using primers and probes constructed using the above criteria combined with the above method.
PCR reactions are typically performed in a 96 well format, however it will be obvious to the skilled artisan that the method is not limited to this format. It is envisioned that the method may also be performed for example, but not limited to, in a single reaction tube (e.g. when multiplexing), “PCR strips” such as 8 well strips or 12 well strips, 384 well plates or 1536 well plates, or any other format, depending on the available volume of sample and the amount of target genes that need to be analyzed.
The method of the invention determines the expression level of six or more genes in a sample, as it is envisioned that preferably for each pathway the expression level of at least three target genes is analyzed, combined with the expression level of at least three reference genes. It is therefore understood that for each additional pathway that is to be analyzed (i.e. for which the activity is to be determined) the expression levels of an additional three or more target genes should be determined in the method. Therefore in order to determine the activity of a single cellular signaling pathway, the expression levels of at least six target genes should be determined, in order to determine the activity of two cellular signaling pathways, the expression levels of at least nine target genes should be determined, in order to determine the activity of three cellular signaling pathways, the expression levels of at least twelve target genes should be determined, etcetera. The method of the invention may be used to determine the activity of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more activities of cellular signaling pathways.
For each cellular signaling pathway individually, the expression levels of three or more target genes are to be determined in the method of the invention, e.g. the expression levels of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more target genes. Similarly, the expression levels of three or more reference genes are to be determined in the method of the invention, e.g. the expression levels of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more reference genes.
When used herein, “simultaneously determining the expression levels” should be interpreted as in a single PCR run, meaning the genes may be amplified and detected in individual wells or containers in the PCR device (e.g. in the separate wells of a 96 well plate, or in separate reaction tubes) or they may also be pooled and amplified in a single reaction (multiplexed), or partially pooled.
The term “sample” as used herein refers to any medium containing nucleotides, preferably RNA, such as but not limited to a medium containing cells, tissue, body fluids, culture medium, or any medium derive from these after further processing steps such as lysis, fixation or isolation of nucleotides, preferably RNA.
It is understood that in order to amplify RNA using PCR technology, first a reverse transcriptase step must be performed. In such a step a reverse transcriptase enzyme is used to generate a complementary DNA form an RNA template. The reverse transcriptase enzyme typically employs a primer sequence which is reverse complementary to a part of the RNA. Generally oligo dT primers may be used for such reaction, or target specific primers. For the method disclosed herein, preferably the reverse primers are used both for the reverse transcriptase reaction and the amplification reaction.
Amplification using DNA primers (polymerase chain reaction) is a technology well known to the skilled person. PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature dependent reactions, such as DNA melting and enzyme driven DNA replication. PCR employs two main reagents, the forward and reverse primers, which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region, and a DNA polymerase.
In method disclosed herein the primers and probes amplify and detect of the expression levels of three or more of the reference genes selected from: ACTB, ALAS1, B2M, EEF1A1 POLR2A, PUM1, RPLP0, TBP, TPT1 and TUBA1B, and wherein the primers and probes further amplify and detect the expression levels of three or more target genes for one or more cellular signaling pathways selected from the group consisting of: AR, ER, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFbeta, WNT, PR, NFkB, JAK-STAT1/2 and JAK-STAT3. Therefore the method may include primers and probes for the amplification and detection of three or more target genes for one, two, three, four, five, six seven, eight, nine, ten, eleven, twelve or more cellular signaling pathways. In a preferred embodiment the one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more, cellular signaling pathways comprise one or more, e.g. one, two, three, four, five, six or seven, cellular signaling pathways selected from the group consisting of the AR, ER, PI3K-FOXO, MAPK-AP1, HH, Notch and TGFbeta cellular signaling pathways. In a more preferred embodiment the one or more, e.g. one, two, three, four, five, six seven, eight, nine, ten, eleven, twelve or more, cellular signaling pathways comprise one or more, e.g. one, two, three or four, cellular signaling pathways selected from the group consisting of the AR, ER, PI3K-FOXO and MAPK-AP1 cellular signaling pathways, and may optionally further comprise one or more, e.g. one two or three cellular signaling pathway selected from the group consisting of HH, Notch and TGFbeta.
In a further preferred embodiment the one or more, e.g. one, two, three, four, five six or seven or more cellular signaling pathways are selected from JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR. In a further preferred embodiment the one or more, e.g. one, two, three, four, five six, seven, eight, nine, ten, eleven or twelve or more cellular signaling pathway comprise one or more, e.g. one, two, three, four, five six or seven or more cellular signaling pathways selected from JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR. These embodiments are particularly useful as the JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR pathways all play a role in immunity, thus a product allowing to determine part or all of these seven signaling pathway activities is a useful tool in e.g. diagnostics of immune related diseases and disorders, such as infection, autoimmune diseases, but also cancer.
Suitable probes types for qPCR are known to the skilled person. For example, the probes may be fluorophore based, such as TaqMan probes. Fluorophore based probes generally work by including a quencher. TaqMan probes consist of a fluorophore covalently attached to the 5′-end of the oligonucleotide probe and a quencher at the 3′-end. The principle relies on the 5′-3′ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. When bound to the probe the fluorophore is quenched, but once cleaved from the probe the distance with the quencher is increased resulting in fluorescence of the fluorophore when excited at the right wavelength.
In a preferred embodiment of the method of the first aspect of the invention, the primers and probes are able to amplify and detect the respective genes under the following reaction conditions:
Therefore, the primers and probes are preferably able to detect the expression level of a single target gen in a medium comprising 50 mM monovalent salt, 3.0 mM divalent salt, preferably the divalent salt being Mg2+ and 0.8 mM dNTP, when the primers are present in a concentration of 400 nM for each forward and reverse primer, and wherein the probe is present in a concentration of 100 nM. It is understood that the primers and probes may work outside these parameters, but that the parameters for designing the primers and probe combinations are designed are such that they are suitable for these reaction conditions.
Preferably the divalent salt is MgSO4 or MgCl2.
In further preferred embodiment of the method of the first aspect of the invention, the primers and probes are able to amplify and detect the respective genes under the following reaction conditions:
In an embodiment of the method of the first aspect of the invention, the method is used to determine one or more cellular signaling pathway activity or activities. Preferably the one or more cellular signaling Pathway activities are selected from the group consisting of AR, ER, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFbeta, WNT, PR, NFkB, JAK-STAT1/2 and JAK-STAT3, even more preferably from the group consisting of AR, ER, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFbeta, JAK-STAT1/2 and NFkB. An especially advantageous aspect of the invention is the ability to determine multiple pathway activities in a sample in a single reaction, therefore preferably two or more, e.g. three, four, five, six or seven or more cellular signaling pathway activities are determined using the method of the invention. Particularly preferred is using the method to determining four or more cellular signaling pathway activities, wherein the four or more cellular signaling pathway activities comprise the ER, AR, PI3K-FOXO and AP1-MAPK cellular signaling pathways. Further particularly preferred is using the method to determining seven or more cellular signaling pathway activities, wherein the seven or more cellular signaling pathway activities comprise the ER, AR, PI3K-FOXO, AP1-MAPK, Notch, HH and TGFbeta cellular signaling pathways.
In a further preferred embodiment the one or more, e.g. one, two, three, four, five six or seven or more cellular signaling pathways are selected from JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR. In a further preferred embodiment the one or more, e.g. one, two, three, four, five six, seven, eight, nine, ten, eleven or twelve or more cellular signaling pathway comprise one or more, e.g. one, two, three, four, five six or seven or more cellular signaling pathways selected from JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR. These embodiments are particularly useful as the JAK-STAT1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch and AR pathways all play a role in immunity, thus a product allowing to determine part or all of these seven signaling pathway activities is a useful tool in e.g. diagnostics of immune related diseases and disorders, such as infection, autoimmune diseases, but also cancer.
It is considered that there are multiple practical applications where the determination of multiple cellular signaling pathway activities may be extremely useful. Although determining pathway activity has been described in the literature by alternative means, known methods are generally not very quantitative and/or easy and/or fast to perform. It is therefore postulated that the presently described solution wherein in a single qPCR reaction multiple cellular signaling pathways activities can be inferred offers extremely useful applications in research and diagnostics, as often multiple cellular signaling pathways are relevant for e.g. making a clinical decision based on pathway activities in a patient sample. The invention provides an easy to perform assay where a quantitative assessment of the activities of e.g. 7 cellular signaling pathways can be determined on a difficult to process sample such as FFPE (Formalin-fixed paraffin embedded) tissue in a matter of 2-3 hours in a single reaction.
When used herein, the terms AP1-MAPK, MAPK-AP1 and MAPK are used interchangeably and refer to the MAPK signaling pathway controlled by the AP1 transcription factor complex. When used herein the terms PI3K-FOXO, FOXO-PI3K and PI3K are used interchangeably and refer to the PI3K signaling pathway, which activity may be determined by taking the inverse of the determined by FOXO pathway activity. When used herein, the terms HH and Hedgehog are used interchangeably and refer to the Hedgehog cellular signaling pathway.
In a preferred embodiment the primers and probes for the ER, AR, PI3K-FOXO, AP1-MAPK, Notch, HH, TGFbeta, JAK-STAT1/2 and NFkB cellular signaling pathways and the reference genes are selected from Tables 1 to 8 and 10 and 11 as described herein below.
In a second aspect the invention relates to an assembly of primers and probe for determining the activity of the ER cellular signaling pathway, wherein the assembly of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the ER cellular signaling pathway, wherein said three or more sets of primers and probes are selected from Table 1 below, wherein each primer and/or probe individually is identical to the corresponding sequence in Table 1 or differs at 1, 2 or 3 positions, wherein said 1, 2 or 3 differences are selected from a single base substitution, a single base deletion or a single base addition.
Tables 1 to 8, 10 and 11 disclose different sets of primers and probes for the different target genes of the referred cellular signaling pathways. The lists are sorted per gene, each gene may have multiple sets of primers and probes, each set of primers and probes consisting of a forward primer, a reverse primer and a probe. Each set is indicated as an “assay”, therefore when used herein, a set of primers and probe refers to a forward primer, a reverse primer and a probe with the same assay name as indicated in one of tables 1 to 8, 10 and 11. The primers and probes are indicated with their respective SEQ ID NO and name (assay), the name being generated according to the following format: [GENE NAME]_[NUMBER], wherein primers and probe with the same name (thus same gene name and number) are part of a single set.
Therefore, the invention further relates to an assembly of primers and probes for determining the activity of the ER cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the ER cellular signaling pathway, wherein said sets of primers and probes are selected from Table 1 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
When used herein, a set of primers and probe refers to set consisting of a forward primer and a reverse primer for amplifying a genomic sequence and a probe for detecting the genomic sequence, where the genomic sequence is a target gene suited for determining the activity of a cellular signaling pathway. The forward and reverse primer and corresponding probe belonging to a single “set” are defined in the “assays” listed in Tables 1 to 8, and thus the primers and probe have an identical assay name when belonging to the same “set”.
When used herein, an assembly of primers and probes refers to multiple (e.g. at least two) sets of primers and probes as defined herein.
In a third aspect the invention further relates to an assembly of primers and probes for determining the activity of the AR cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the AR cellular signaling pathway, wherein said sets of primers and probes are selected from Table 2 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a fourth aspect the invention further relates to an assembly of primers and probes for determining the activity of the PI3K-FOXO cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the PI3K-FOXO cellular signaling pathway, wherein said sets of primers and probes are selected from Table 3 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a fifth aspect the invention further relates to an assembly of primers and probes for determining the activity of the AP1-MAPK cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the AP1-MAPK cellular signaling pathway, wherein said sets of primers and probes are selected from Table 4 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a sixth aspect the invention further relates to an assembly of primers and probes for determining the activity of the Notch cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the Notch cellular signaling pathway, wherein said sets of primers and probes are selected from Table 5 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a seventh aspect the invention further relates to an assembly of primers and probes for determining the activity of the HH cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the HH cellular signaling pathway, wherein said sets of primers and probes are selected from Table 6 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a eight aspect the invention further relates to an assembly of primers and probes for determining the activity of the TGFbeta cellular signaling pathway, wherein the set of primers and probes comprises at least three sets of primers or probes for determining the expression level of three or more target genes of the TGFbeta cellular signaling pathway, wherein said sets of primers and probes are selected from Table 7 wherein the forward primer, the reverse primer and the probe have the same assay name; and
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 8 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
In a ninth aspect the invention further relates to an assembly of primers and probes according to any one of the preceding claims further comprising primers and probes for determining the activity of the JAK-STAT1/2 cellular signaling pathway, wherein the assembly of primers and probes comprises three or more sets of primers or probes for determining the expression level of three or more target genes of the JAK-STAT1/2 cellular signaling pathway, wherein said three or more sets of primers and probes are selected from Table 10 of the description,
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 10 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a tenth aspect the invention relates to an assembly of primers and probes according to any one of the preceding claims further comprising primers and probes for determining the activity of the NFkB cellular signaling pathway, wherein the assembly of primers and probes comprises three or more sets of primers or probes for determining the expression level of three or more target genes of the NFkB cellular signaling pathway, wherein said three or more sets of primers and probes are selected from Table 11 of the description,
Preferably the assembly further comprises three or more sets of primers and probes for determining the expression levels of three or more reference genes, wherein said sets of primers and probes are selected from Table 11 wherein the forward primer, the reverse primer and the probe have the same assay name; and
It is recognized that the PCR reaction is tolerant to a few, e.g. 1, 2 or 3, mismatches in the primer or probe sequences and still allows amplification and detection of the target gene expression level. Therefore, up to 3 mismatches may be allowed in the sequences of the primers and probes each. A mismatch or difference at a position of a sequence when used herein refers to a single base substitution, a single base deletion or a single base insertion with respect to the reference sequence (e.g. the respective SEQ ID NO). It is recognized that the PCR reaction is general more tolerant to a substitution as compared to a deletion or insertion in the primers or probes, therefore preferably the mismatch or difference at a position of a sequence is a single base substitution, although a deletion or insertion ay be desirable to account for genetic variation in the target DNA of the sample.
It is understood that avoiding mismatches is preferable for optimal amplification and detection of the target gene. Therefore preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 or 2 positions, more preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO or differs at 1 position, most preferably each primer and/or probe individually is identical to the corresponding SEQ ID NO.
The assembly may further comprise one or more, e.g. one, two, three, four, five, six, seven, eight or more, additional assemblies of primers and probes as defined herein.
In a particularly preferred embodiment, the invention relates to an assembly of primers and probe for determining the activity of the AR cellular signaling pathway, and optionally one or more additional cellular signaling pathway, wherein the assembly of primers and probes comprises three or more sets of primers or probes for determining the expression level of three or more target genes of the AR cellular signaling pathway, wherein said three or more sets of primers and probes are selected from Table 2 of the description,
In an embodiment of the assembly according the second to the tenth aspect of the invention, all of the primers and probes in the three or more sets of primers and probes in the assembly are identical to the corresponding sequences according to Tables 1 to 7, 10 and 11 or the sequences represented by the indicated SEQ ID Nos.
In a eleventh aspect the invention relates to a kit of parts for determining the expression levels for a plurality of genes, the kit comprising primers and probes for the amplification and detection of the expression levels of the plurality of genes, wherein the kit comprises primers and probes are as defined in the first aspect of the invention or an assembly of primers and probes as defined in any one of the second to the tenth aspect of the invention, wherein the kit further comprises primers and probes for the amplification and detection of three or more of the reference genes selected from ACTB, ALAS1, B2M, EEF1A1 POLR2A, PUM1, RPLP0, TBP, TPT1 and TUBA1B. Preferably said three or more sets of primers and probes are selected from Table 8 of the description, and wherein each primer and/or probe individually has the listed nucleotide sequence identified by the corresponding SEQ ID NO or has a sequence that differs at 1, 2 or 3 positions, wherein said 1, 2 or 3 differences are individually selected from a single base substitution, a single base deletion or a single base addition.
Preferably the kit is suitable for determining the activity of a cellular signaling pathway. Therefore the kit preferably comprises at least three sets of primers which are suitable for the amplification and detection of the expression levels of at least three target genes of a cellular signaling pathway as described in the first to the eight aspect of the invention. Preferably the cellular signaling pathway is selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, WNT, HH, PR, TGFbeta, NFkB, STAT1/2, STAT3, and Notch. More preferably the kit comprises three or more sets of primers and probes, e.g. three, four, five, six, seven, eight, nine, ten, eleven or twelve or more, as described herein each for the detection of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, pathway activities selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, WNT, HH, PR, TGFbeta, NFkB, STAT1/2, STAT3, and Notch, more preferably selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, Notch, HH and TGFbeta. Preferably said one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, pathway activities selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, WNT, HH, PR, TGFbeta, NFkB, STAT1/2, STAT3, and Notch comprise one or more, e.g. one, two, three, four, five, six or seven, eight or nine pathways selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, Notch, HH, TGFbeta, JAK-STAT1/2 and NFkB. Preferably said one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, pathway activities selected from the group consisting of ER, AR, PI3K-FOXO, MAPK-AP1, WNT, HH, PR, TGFbeta, NFkB, STAT1/2, STAT3, and Notch comprise one or more, e.g. one, two, three or four, pathways selected from the group consisting of ER, AR, PI3K-FOXO and MAPK-AP1, or comprises one or more, e.g. one, two, three, four, five, six or seven pathways selected from the groups consisting of ER, AR, PI3K-FOXO, MAPK-AP1, Notch, HH, TGFbeta, or comprises one or more, e.g. one, two, three, four, five, six or seven pathways selected from the groups consisting of JAK-STAT 1/2, NFkB, TGFbeta, PI3K-FOXO, MAPK-AP1, Notch en AR.
Optionally the primers and probes for the amplification and detection of the reference genes are selected from Table 8 below, although it is understood that other sets of primers and probes may be used, provided the primers and probes are suitable at the reaction conditions described herein above.
The kit may optionally further comprise one or more of a polymerase enzyme, a reverse transcriptase enzyme, a suitable buffer and a container for performing the PCR reaction. Suitable containers may be reaction tubes, PCR strips such as 8 well or 12 well strips or multiwell plates, also known as microwell plates, microtiter plates or microplates, such as 6, 12, 24, 48, 96, 384, or 1536 well plates, or any other container which can be used in a thermal cycler, preferably a thermal cycler with fluorescence readout capability.
In an twelfth aspect the invention relates to the use of the primers and probes as defined in the first aspect or the assembly of primers and probes as defined in the second to the tenth aspect or the kit as defined in eleventh aspect for determining the cellular signaling pathway activity for one or more cellular signaling pathways selected from the group consisting of: ER, AR, PI3K-FOXO, MAPK-AP1, WNT, HH, PR, TGFbeta, NFkB, STAT1/2, STAT3, and Notch.
In a thirteenth aspect the invention relates to the use of a set of three or more primers and probes to determine the expression levels of three or more target genes of a cellular signaling pathway, wherein the primers and probe combinations are as defined in the second to the eleventh aspect, and
In a fourteenth aspect the invention relates to a method for designing primers and probes for the detection of the expression levels of target genes of a cellular signaling pathway suitable for determining the activity of said cellular signaling pathway, said cellular pathway preferably being one or more, for example on, two, three, four, five six, seven, eight or nine, cellular signaling pathways selected from the groups consisting of ER, AR, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFBeta, JAK-STAT1/2 and NFkB, the method comprising:
Primers designed according t this method are useful as they can be used in an assay for determining one or more cellular signaling pathway activities. Preferably the primers and probes are able to detect the expression level of a target gene under the following reaction conditions:
Preferably the primers and probes are able to amplify and detect the respective genes under the following reaction conditions:
In a fifteenth embodiment the invention relates to a method of determining the a cellular signaling pathway activity selected from ER, AR, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFBeta, JAK-STAT1/2 and NFkB and optionally one or more additional cellular signaling pathway activity or activities, preferably said one or more additional cellular signaling pathway activities are also selected from ER, AR, PI3K-FOXO, MAPK-AP1, HH, Notch, TGFBeta, JAK-STAT1/2 and NFkB, by simultaneously determining the expression level of six or more genes in a sample, the method comprising simultaneously amplifying six or more gene products using a polymerase chain reaction to generate a plurality of amplification products, followed by the detection of the plurality of amplification products using a plurality of probes,
This application describes several preferred embodiments. Modifications and alterations may occur to others upon reading and understanding the preceding detailed description. It is intended that the application is construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.
It shall be understood that the assemblies of primers and probes, the kit, the different uses, the method for designing primers and probes, and the method for determining AR activity have similar and/or identical preferred embodiments, in particular, as defined in the dependent claims.
In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.
A single unit or device may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
It shall be understood that a preferred embodiment of the present invention can also be any combination of the dependent claims or above embodiments with the respective independent claim.
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.
Primers and probes for target genes of the following cellular signaling pathways were designed as described herein:
The designed primers and probes are listed below in Tables 1 to 7. Further, the same principle was used to design primers and probes to detect the expression levels of several reference genes, which are listed below in Table 8. All primers and probes were validated using the following conditions:
All sets of primers and probes were found to amplify and detect the expression levels of the desired target genes at the indicated reaction conditions.
All primers and probes were validated under the following conditions:
A RT reaction at 50° Celsius for 30 minutes (using the reverse primer) in order to synthesize cDNA, followed by a 5 minute denaturation step at 95° Celsius, followed by 45 cycli of a 15 second denaturation step at 95° Celsius and a 30 second Elongation step at 60° Celsius, the reaction was performed on a qPCR device with fluorescence readout.
Melt curves were determined in the range of 60° C. to 95° C.
Some exemplary data sets for validated genes are provided below, although it is noted that all genes described below were validated.
Additional information is provided for the following assays: ABCC4_2, GREB1_2 and GADD45A_2, which comprise primers and probes for the amplification and detection of the respective genes ABCC4, GREB1 and GADD45A. Assay ABCC4_2 results in an amplification product of 82 nucleotides spanning an 11594 nucleotide intron. Assay GREB1_2 results in an amplification product of 105 nucleotides spanning an 9696 nucleotide intron. Assay GADD45A_2 results in an amplification product of 82 nucleotides spanning an 1037 nucleotide intron.
Some general information on the assays is listed in Table 9 below.
In order to validate the assays, they are tested on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. The assays were tested on in vitro RNA, cell culture isolate RNA and RNA isolated from a formalin fixed paraffin embedded (FFPE) sample, to test their suitability. For all tests the probes were tested on decreasing amounts of material (RNA) in order to test their efficiency.
Real-time probe fluorescence curves were generated for all tests performed, as well temperature melt curves over a range of 60 to 95 degrees Celsius. These data are depicting in
Most probes and conditions resulted in a probe efficiency of over 90%, indicating the probes are suitable for quantitative measurements of target gene expression levels.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20193288.6 | Aug 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/073469 | 8/25/2021 | WO |
