Troubleshooting issues:
In RT-PCR, we have had trouble with our usual amplifications of 18S using SYBR green primers. We have tried multiple, already tested and confirmed sets of primers, and multiple sets of samples. We thought this may have been an issues with cDNA synthesis, however when testing other targets with SYBR green primers, the amplification was as expected.
In 18S runs, with ~40 samples, we have a range from 20-45 cycles for Ct values while the other targets are within a narrow (expected) range of 3-5 cycles. The problem has appeared in a number of other experimental models in mouse tissue though the variability does not occur in C2C12s -- only harvested, mouse skeletal muscle. The Tm Peaks look normal. We have only just switched to SYBR for our endogenous control. Previously, TAQMAN probes for 18S were used and they worked fine.
Any suggestions/ideas? Why would this gene that is hardly ever altered, even slightly, show dramatic variances in expression?
cDNA was synthesized for all RNA samples by DNAse treating 1ug of RNA, synthesized using qScript cDNA master mix according to manufacturer protocol, and RNAse H treatment afterwards. Serial dilutions were made to get 1:100 cDNA for RT-PCR. This has been a standard protocol of the lab and has worked successfully in the past. We thought this process was the issue until we saw targets that amplified normally. This told me that the cDNA synthesis process worked successfully as expected.
You did not answer the question: did you use a mix of random primers or did you use oligo (dT) primer?
I have looked at the manufacturer's site and they say they provide a "unique blend" of oligo (dT) and random primers. I suggest you to do the experiment again only with random primers.
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