Help! DNA contamination in RNA and RT-PCR. - (Nov/18/2002 )
i am trying to extract the total RNA from the Gram negative bacteria to do RT-PCR.i use Qiagen Kit, RNeasy mini kit to extract the RNA and i always do DNase digestion after the last elusion step. when i check the quality of the RNA, the OD260/OD280 is arroung 2.0 and it seems that the quality of RNA is good. but, in the later RT-PCR step, the chromosome DNA contamination is still there. Do you have any good suggestion about that?
is it possible that in prokaryotic cells, the replication and transcription is always coupled together, esp. for the house-keeping genes, so the DNA and RNA is tied so tightly that they are not easy to separate in the extraction steps? the result will be that when you want to do RT-PCR for the house-keeping gene, the corresponding chromosome DNA will always be there to cause the contamination. is this guess likely?
I'm using the same kit as you're, and i've had no problems whatsoever. I use the RNA in Northerns and RT-PCR and I see no contamination with DNA. Maybe you should check your DNase or try a new batch or elongate the DNA digestion step....