I am trying to transfect RNA into cells. My transfection experiments seem successful as my luciferase readouts are to the tune of 100000 for 10 ul of cell lysate. When I obtain total RNA after transfection and do a reverse transcription, the transcription doesnt work which is fascinating. Has anyone come across this problem?
Hi Anjali!
I'm not ready to answer you question, but if you use lipocationic transfection, I strongly recomment you to read this paper: http://rnajournal.cshlp.org/content/12/10/1790.full.pdf+html.
As you will see, the way you are going to may be wrong...
Sergey.
What does the RNA you transfect look like and how do you obtain it? Is it just in vitro transcribed? Does it have a poly A tail, and what oligo are you using for the reverse transcription? My first guess would be that the poly A tail might be missing and this is why you can't reverse transcribe your RNA (if it's a oligodT primer for RT).
Oh I am sorry Sascha and Sergey for the insufficient information given. I make the RNA invitro and it has a T7 promoter with strong start codon and a poly A tail. The RT primer is again custom made (PAGE purified) and recognizes a region within the 5' leader sequence to make the reverse transcription product . I have done a control experiment with RNA on its own and reverse transcription worked perfectly.
@Sergey: Thanks, I will look into this paper and see what could be wrong as per your suggestion.
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