Methods and reagents that specifically detect, distinguish and quantify IFN-λ2 mRNA from IFN-λ3 mRNA in humans
The present invention provides a method of specifically detecting IFN-λ2 mRNA or IFN-λ3 mRNA. There is provided a qRT-PCR method specifically detecting, discriminating and quantifying IFN-λ2 and IFN-λ3 mRNA in a biological sample obtained from a human. There is provided qRT-PCR methods and primers and probes that specifically detect IFN-λ2 mRNA but not IFN-λ3 mRNA and vice versa in humans in order to detect, quantify and discriminate IFN-λ2 mRNA and IFN-λ3 mRNA.
1. A method of quantifying IFN-λ2 mRNA but not IFN-λ3 mRNA in a biological sample in humans, comprising the steps of:
a) obtaining a biological sample from a human, said biological sample is suspected of having IFN-λ2 mRNA;
b) isolating mRNA from said biological sample;
c) performing a reverse transcription reaction to convert said mRNA to cDNA;
d) performing a qRT-PCR on said cDNA using an IFN-λ2-specific primer set, said IFN-λ2 specific primer set containing a forward primer and a reverse primer to yield an amplification product, wherein said forward primer consists of SEQ ID NO: 3, and said reverse primer consists of SEQ ID NO: 4, wherein said IFN-λ2-specific primer set amplifies under conditions suitable for amplification of nucleic acid sequence of IFN-λ2 mRNA and not IFN-λ3 mRNA, wherein said qRT-PCR is specific for IFN-λ2 but not IFN-λ3 with low cross-reactivity as evidenced by generation of one single peak in a dissociation curve analysis; and
e) quantifying said amplification product to determine the expression level of IFN-λ2 mRNA.
2. The method of claim 1 , wherein said biological sample is a dendritic cell.
3. The method of claim 1 , wherein said biological sample is an airway epithelial cell.
4. The method of claim 1 , wherein said biological sample is a colon cell.
5. The method of claim 1 , wherein said biological sample is a liver cell.
6. The method of claim 1 , wherein said qRT-PCR step is performed using an annealing temperature of 60° C.
7. A method of quantifying IFN-λ3 mRNA but not IFN-α2 mRNA in a biological sample in humans, comprising the steps of:
a) obtaining a biological sample from a human, said biological sample is suspected of having IFN-λ3 mRNA;
b) isolating mRNA from said biological sample;
c) performing a reverse transcription reaction to convert said mRNA to cDNA;
d) performing a qRT-PCR on said cDNA using an IFN-λ3-specific primer set, said IFN-λ3 specific primer set containing a forward primer and a reverse primer, wherein said forward primer consists of SEQ ID NO: 10, and said reverse primer consists of SEQ ID NO: 11 to yield an amplification product, wherein said IFN-λ3-specific primer set amplifies under conditions suitable for amplification of nucleic acid sequence of IFN-λ3 mRNA and not IFN-λ2 mRNA, wherein said qRT-PCR is specific for IFN-λ3 but not IFN-λ2 low cross-reactivity as evidenced by generation of one single peak in a dissociation curve analysis; and
e) quantifying said amplification product to determine the expression level of the IFN-λ3 mRNA.
8. The method of claim 7 , wherein said biological sample is a dendritic cell.
9. The method of claim 7 , wherein said biological sample is an airway epithelial cell.
10. The method of claim 7 , wherein said biological sample is a colon cell.
11. The method of claim 7 , wherein said biological sample is a liver cell.
12. The method of claim 1 , wherein said qRT-PCR step is performed using an annealing temperature of 60° C.