I had isloated total RNA from frozen tissues using the Qiagen RNeasy kit.
My goal was to use this to make cDNA and then perform regular PCR in order to determine which isoforms of my protein were being expressed. For this I was using the Invitrogen - 'Superscript III One Step RT-PCR with Platinum Taq polymerase'.
I had designed gene specific primers but when I carried out the RT-PCR, I ended up getting multiple bands besides the band of the predicted size (which was also the brightest).
So then I decided to check for genomic DNA contamination by carrying out the same RT-PCR, except instead of adding the superscript III enzyme (for reverse transcription), I added only Taq DNA polymerase - ideally I should not see any PCR product, but in this case I did see 2 -3 bands - which I presume means that my primers were primming off of the genomic DNA.
I dont have much of RNA sample left, about 8microliters (of conc ~ 50ng/ microliter ) and with whatever I have left I just want to do a quick and dirty analysis to know if this is the right direction to pursue.
MY QUESTION - Can I just add DNase I to my RNA sample, incubate it for a while at room temp, maybe inactivate the DNase I at 65C AND THEN DIRECTLY USE THAT SAME SAMPLE TO PROCEED ONTO RT-PCR using the Invitrogen kit???????????
help - RT-PCR