Hello!!
Can anyone tell what could be the reason to get completely opposite Cts value in 2 different qPCR with same qPCR components?
I was testing a certain primer on different tissues, first time i TurboDNAse treated on 1ug of extracted RNA then made 10ul of CDNA with 0.5ug treated RNA and run qPCR 10ul of reaction
I got CT values pretty high (28-38) except in one tissue where it was around 22Ct.
Second time i wanted to run with reference gene, so i remade cDNA with 0.5ug but with 20ul of cDNA reaction and ran with a bit different cycling conditions.
And i got pretty low CT around 8-12 except the same tissue which had 22 Ct in previous run had Ct 33. So, it was just opposite results.
Can anyone has any idea why it can be?
Regards
Did you use the same batch of RNA both times? It sounds like something is very wrong with either your RT reaction setup or your qPCR reactions. What primer type are you using for your reverse transcription? For your qPCRs, is this SYBR green or a probe-based reaction, and can you share the amplification curves and any melt curve data that you have?
Laura Leighton , thank you for your answer. I figured out the issue, i was using wrong cycling conditions when i repeated the run :D
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