I am trying to amplify cytokine genes in cDNA derived from macrophages but unable to get any amplification using comparative delta Ct experiment. Cross checked mRNA concentration and purity which is fine but still cant get Ct values. Please suggest possible reasons and ways to troubleshoot
what about the reference gene used in ur comparative experiment?
Could u see any amplification in ur entire setup?
If u have time, start checking everything right from ur instrument (telling u, thorough understanding & these checks will make u understand deep about everything on this matter and makes u master)
Yes I am getting amplification in GAPDH internal control but not in test genes, can it be probably a primer issue?
apparently!
Tried amplifying ur target with that set on routine PCR? verified for amplification?
Is this set of primers having any abnormal Ta, with which u cant club them with GAPDH set?
Maybe the genes you are interested in are poorly expressed in your samples so you do not reach the threshold value in the number of cycles you are using for your RT-PCR. You might try to start from a more concentrated sample of cDNA so the number of copies you start from is higher.
if you primers work, you should include positive control samples such as LPS stimulated macrophages. these samples should have high expression of many inflammatory genes.
you should standardize your experiment taking different concentration of cDNA (from low concentration to high) and Primers.
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