I am new to doing RT-PCR. I have done a bit of standardization. But stuck with one problem which I dont understand where could the problem possibly be....
I use Trizol (TaKaRa) for RNA isolation followed by cDNA synthesis (verso kit, Thermo).
one problem I am facing is DNA contamination for which I have reduced the original culture for RNA isolation so as to get as little DNA as possible. Then I treat the sample with DNAse (37 degree C for 15 mins) followed by LiCl precipitation (7M) and three volumes of Ethanol for 3 hrs at -20 degree C.
For cDNA synthesis, I have tried different temperatures : 42 degree C for 1 hour and 50 degree C and 55 degree C for 30 minutes
The whole cycle for cDNA synthesis:
RNA (200-400ng) random hexamers and water at 65 degree C for 5 mins, add other reagents and incubation at 42/ 50/ 55 degree C for ihr/ 30 minutes, followed by 94 degree C for 2 mins.
when I do RT-PCR using cDNA, there is no difference in Ct values between NRTC control and experimental set when house-keeping genes gyrB and 16S are used. The melt curves seem to be okay
I have done verification of these primers with PCR and I get single sharp band of expected size.
Can anyone help please?? Is there a problem with cDNA synthesis....what could be the possible reason for no difference in the Ct values??