Troubleshooting reverse transcriptase-PCR - (Nov/26/2010 )

Hi to all

I am a new member here...I hope someone can give me advices for my problem..

I have been struggling with an RT-PCR for a few months now. Starting material is total RNA extracted from Vicia faba leaves using RNeasy Qiagen kit. I have no bands in the gel after doing my PCR. I have tried RT with random hexamers as well as oligo dT primers. I have tried all possible combinations of MgCl2, dNTP and primer concentrations. Still the amplification is none. I have tried two PCR master mixes (promega and fermentas) which Don't work at all. Tried varying denaturing, annealing and extension temps and times, tried touch down.. I have checked my RNA on agarose gel...and I can see clearly 28S and 18S bands in the ratio 2:1.. Any suggestions are welcome, Somebody please help me ..............

Hello everyone...

I see that no one replied to my problem...

I will just add some hints..I hope it can help

Till now I am sure that my starting material (total RNA) is fine...I don't know if the problem is in the RT reaction or in the PCR process...I always do two step RT-PCR...First I do RT reaction...denaturing of 4 ul RNA with 1ul dT primer at 70C for 5 min..then quickly on ice..then making the RT mix using 1 ul RT enzyme and the buffer and nuclease free water in a total volume of 20 ul...the RT reaction lasts for 80 min. with 25C for 5 min, 42C for 60 min then inactivation at 70C for 15 min. After that I proceed with PCR using random hexamers along with oligo dT primer...with no amplification results at all for two months now...

Sometimes I used to run a volume from the product of RT reaction on 1% agarose gel ...there used to be a smear with a thick band at the top of the gel near the well..when I tried running a DNA marker along with this thick band..it turned out to be about 10.000 bp which is impossible to be the product of RT...I thought it was contamination...revised all steps and repeated the procedures..with the same band appearing after RT reaction..but I don't know where is the problem?!...even if I am having this so big band it doesn't amplify in PCR reactions...Please help me and tell me what is wrong...

Why would you expect a sharp band with random hexamers and poly-A primers? You won't get a specific product without a gene-specific primer. With these primers, you will get a smear, which is exactly what it sounds as if you are getting. I'm guessing your reactions are working fine, and that with a gene specific primer you would be off and running. Ideally, you should have a positive control -- a primer known to work in your organism, as well as the specific primer for the gene you are looking for.

Hi,
Have you check any housekeeping gene expression after reverse transcription PCR (RT-PCR)? This can make sure your RT-PCR was successful.
Alternatively, maybe you can try to clean up your RNA again with kit and see how it goes.

Best regards,

Lee