problem in isolation of HCV RNA and then RT-PCR - RT-PCR, HCV virial RNA (Jun/10/2008 )
I have a problem now and hope someone here can give me an advice.
my purpose is to get the core region of HCV. so I disigned a pair of primer matching to core region. today, I isolated HCV virus RNA by using QIAamp virus RNA mini kit from 400 ul of serum stored at -80 degree for 3 years. then did cDNA transcription by using SuperScript first strand synthesis system for RT-PCR from invitrogen. finally, PCR was done.
Tm of my specific primer are 75 and 67 degree. but since the primer for control Reactions was suggested a Tm at 58 degree, so I set the annealing temperature at 58 degree for 30 s. extension degree at 72 degree for 1 min. 35 cycles.
finally, I got an expected band for control reaction (transcription by using control RNA, then PCR). but got smear for my target product.
do you think that the failure is due to low annealing temperature, or I failed from the step of RNA isolation?
I wondered whether 400 ul of serum is enough for isolation of virus RNA. but the patient is clearly HCV positive. I will try higher annealing temperature tomorrow. but at mean time, I wish someone can teach me what should be paid more attention for this experiemtn. especially, I do not have expeirence of isolating virsu RNA. anything special with this?
thank you in advance!!
The problem you have to solve is your specific primer, Tm seems too high, try to re-design primer to make its Tm close to your control. To make sure the successful isolation, you can run PCR only with your HCV primer at high annealing temperature.