band in -ve rtPCR - (Aug/22/2008 )

Hi all,
I transfected some HeLa cells with a construct encoding a protein of my interest (rtTA), then extracted RNA and make cDNA.

I use the same cDNA in the same termoblock with 2 different master mix (everything the same but the primer):

GAPDH primer work fine and band only in RT+ cDNA
rtTA primer give me a band as well in -ve cDNA

and..water in PCR using GAPDH primer was clean, but one in rtTA primer had a faint band

I used two different kit to make cDNA and bought 2 set of primer for rtTA with the same result

I'm going to increase the Tm for rtTA primers..any other suggestion??

Thanks in advance
ale

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How are you extracting the RNA? I recommend using the Qiagen RNeasy kit which is a column purification technique. I also recommend you homogenize your cells using the QIAshredder from Qiagen as well prior to purification. Also, you would want to perform a DNaseI digestion. The Qiagen kit allows you to perform the DNase digestion right on the column prior to elution of the RNA.

If this doesn't work, there is one other thing you can do that did work well for me. I homogenized my cells first using the QIAshredder, then I purified the RNA on the spin column but didn't do the on-column DNAse digestion. After I eluted my RNA (which probably still had contaminating DNA in the eluate) I did a restriction digestion with DpnI. DpnI only cuts methylated DNA produced in the cell (not PCR) and will not harm the RNA. After the DNA was cut up in a zillion little pieces, I repurified the RNA on a new spin column, and this time performed a DNaseI digestion.

I hope this helps!

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Do your rtTA primers span an intron enabling you to differentiate between cDNA and genomic DNA? If not, then this could be genomic DNA contamination. It also appears that you have some general contamination (because your water band is positive). Start with all new solutions, use barrier tips, and try to use separate pipettors for setting up the PCR and for loading your gel. You could try to increase your annealing temp to get rid of the top band, but I wouldn't worry too much about it for now- you need to get rid of your contamination first.

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