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maybe someone has a clue. I measured a target gene against five reference genes in a SYBR green assay. I had liver samples of mice and made two seperate tRNA extractions and RTs per sample to have independend replicates of each sample (A and
. Teh problem that now occured is that I have absolutly no reproduceability between the A and B samples (see attached picture). First I analysed the A and B sample in different runs, but the differences between A and B was even confirmend when I re-run them in the same qPCR run. Therefore it could not be inter-run variability. When I had a closer look, I realized that the Ct-values for the five reference genes are almost the same for A and B but that the target gene produces the variance. As this is even reproducable for A and B it seems like there has been a different RT efficiency for my target gene whereas the reference genes where not affected by this. I can not explain how this should happen, as I used dT18 primers for the RT.I can not explain it to me, but it seems to be the only explaination that is left for me. has anyone ever observed something like this or can explain it to me?
I should add that most of the animals are heterozygous for the target gene. The PCR runs where all fine, nice efficiencies, single melting peaks etc..
Thanks to everyone who find's the time to help me.
Jan
p.s. the picture shows the different relative expression values of A and B for each sample
Attached File(s)
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Bild1.jpg (66.91K)
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