I am performing qPCR to quantify cytokine expression in human peripheral blood mononuclear cells. I have found it very difficult to get consistent results when assaying for IL-17A. Currently I am using RotorGene SYBR Green PCR mix, with QIAGEN Quantitiect primers, as well as a set of custom primers adapted from a paper. My cDNA is prepared from RNA isolated using QIAGEN RNeasy kits, and reverse transcribed using SuperScript II RT and Oligo(dT) priming. However I always get a very short range on the standard curve in which there is consistency between replicates. When assaying my study samples there will often be failed reactions.
Looking for advice on how I could optimise my reactions, or recommendations for primers (either for SYBR Green based qPCR, or TaqMan Primers and probes) that have been validated on low abundance transcripts, that I could try for my work
Jack Greenhouse Hi Jack,
Yes I am using GAPDH as the housekeeping gene. I am thinking of now switching from SYBR Green to TaqMan in the hope that this would improve results.
Yes, we initially tried on cDNA from stimuated Jurkat cells hoping there would be detectable IL-17 production, however this did not work. I did manage to obtain a cDNA sample from a colleague from a patient with infection known to induce high Th17 response and IL-17, the primers and assay worked fine on this sample leading me to the conclusion that it must be down to low abundance in my other study samples causing the problems. We did also buy a commercial total human reference cDNA sample, and a human spleen reference cDNA sample, both of these had the same problem with unreliable amplification of any product, and inconsistency between replicates.
It was suggested that I could try a pre-amplification of the cDNA before qPCR however I have not attempted that yet unless I cannot resolve the issue with a simple change to the protocol.
Thanks for your response
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