gDNA contamination in RT-PCR - (Jan/19/2003 )
I know this seems like a long request, but I could really use someones advice here!
After reverse transcription of my RNA (which has been isolated from cells using a Qiagen kit) I have have been using the cDNA in several PCR's (9 in total). The negative controls that I have been using include a neg. RT, no RNA (pos. RT) and NFW control. To date I have had no product for any of the PCR's in my no RNA or NFW controls, which is great.
My problem is that for a couple of the PCR's I have been getting a product in the neg. RT lane. At first I didn't think that this was gDNA as the size of a gDNA band sould be larger (as the primers do span several introns). But I have read that this may be due to pseudogenes. The PCR's that I am having trouble with are my housekeeping genes (GAPDH and Beta-actin).
If the neg. RT lanes are neg. for my other PCR's, from the same reverse transcription reactions, are these results valid? Also if I reduce the number of cycles from 35 to 25 I still get product in my pos. RT lane but not my neg. RT lane, but is this valid?
Thanks so much for the help
It is possible that your reagents have got contaminated. Do you use a hood to set up your reactions? Also, do you change your gloves in between ? That happened with me once.
I don't think it's the pseudogenes acting in your case even though youare using Gapdh and beta actin.
I think it's just contamination. It happened to me a while back too where my neg was not negative. I made a fresh batch of primers and made sure i used RNase free water.
Try it, good luck.