DNA free RNA for PCR - (Nov/03/2006 )
When I did RT-PCR (35cycles) for my gene of interest I did get a very very faint band and nothing in -ve control. The gene is expected to be expressed in very low amounts. WHat I tried was to use the pcr product (ie reaction mix after first PCR of 35 cycles) as template for one more PCR providing all ingredients for next 30 cycles. What I saw was moderate expected band for sample and a faint band for -ve control (No RT). So the doubt I have is irrespective of using good colums for RNA extraction or enough DNase treatments still some genomic DNA might remain which is so low that PCR can amplify only after a second round (total 65 cycles!). Is there any limit for number of cycles till which if negative control is clean we can say that RNA is free of genomic DNA.
Thanks in advance for inputs. (check the picture attached)
My PI says that if you get a band after 30 cycles, it's not significant. This goes for negative control AND cDNA template...
My opinion...he's too fussy. If I see a band after 30 cycles I think it's acceptable to say that the gene is expressed, if you have no band in the negative control. But after 65 cycles... I wouldn't rely on any band coming up.
I forgot to add one comment that this second round of pcr was my PI's suggestion
I agree with Jou
what is the purpose of your PCR? are you trying to make a clone? is that why you need so much?
No Aimee, we are interested in checking the presence of a gene which is expressed only by about 0.1% of the cells present in the culture system. We can not isolate those particular cells but of the whole structure I have isolated RNA and made cDnA . So my aim is ti check whether a gene is expressed which will be only in those subset of cells. So transcripts amount will be too low. Thats why this attempt!
just wondered whether there are special circumstances that keep you from doing a nested PCR?
Doing so, you should be able to amplify your gene product and even increase the specifity. In my experience, it is necessary to exchange at least one primer (hemi-nested) or both primers, to get reliable results. 60 cycles with the same primers will allways bring some bands...
well the pimers should be ordered in separate exons and the genomic DNA wouldn't serve as a template...
so if the band is at the expected size, it's probably not genomic DNA.