gDNA contamination in RT-PCR - (Jan/19/2003 )

Hi All,

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
Kirsty
:-)

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.