Gel and Ct-values relationship: is there a probe problem? - Bands on gel don't fit my expectations... (Sep/29/2006 )
again a qPCR problem...
I run qRT-PCR in Abi prism 7700 using TaqMan technology (fluorescent probe labelled with 5'-FAM and 3'Black Hole Quencher from Eurogentec) to quantify different extra cellular matrix proteins mRNAs in fibroblasts populations.
I'm optimizing a couple of new primers and probes yet, with cDNA from control fibroblast populations. It worked very well this way before with other genes, but now I have problems with these ones.
So I ran a 2% agarose gel after real-time PCR to see what happens...
Surprise! I explain:
- For 2 genes (let's say A & B ), I get normal Ct values (I say normal because in the range of what I expect due to HK gene results) but no band on agarose gel.
- For 1 gene ©, I get high Ct values (>40) and no band on gel
- And for another gene (D), I get high Ct value (approx. 37-39) and bands are visible on gel
I get mad.... Has anyone already experienced such a case? Does anything go wrong with primers &/or probes? (which I designed myself, I precise)
For gene C, it is fully possible that my population of cell does simply not express it. Ok.
But is there a problem of probe with gene D? What's the problem with A and B??
I hope some specialists of yours can enlighten me...
what do you mean by 'normal' Ct's?
anything as high as 40 is not likely to be good
for your C and D I would say that you either have very low expression or you are seeing late-cycle contaminants
for A and B, I have no idea why you see no bands. that makes no sense at all, if you are getting Ct's in a believable range
how reproducible are these results? and, can you post a pic?
thanks for answering. I cannot post a pic now because my picture is so bad that I didn't scan it (quite old devices in my lab ). The bands are very weak indeed.
Normally I admit that Ct higher than 38 is not good. What I call normal Ct is in the range of 25-35 cycles.
On my gel I loaded two RT-PCR samples out of 3. I use to perform reverse transcription of one single RNA sample in triplicate, and then to use each of these triplicates for qPCR.
I did this peculiar test only once because I got high Ct values while trying to optimize primers and probe. And I was wondering how to interprete that. It is good to have specialist to ask for here on this forum, because my referee is a protein biochemistry- specialist, and has no experience with this kind of stuff.
Thanks if someone has more ideas...