RT-PCR question - genomic DNA amplified with RNA (Mar/08/2006 )
My lab is having a problem with our RT-PCR. We are by no means experts in doing RT-PCR, just a few of us who have done it occasionally in the past.
Here is our problem. We have Hela cells transfected with a cDNA. We want to look at RNA levels for that same cDNA (to look for expression within the cell). We collect the cells, run them through Qiagen RNeasy kit with the optional DNASE I step. When we run RT-PCR using a "one-step" kit we see a band of correct size, however, if we use the same template and run regular PCR with Taq we also see a band of the correct size. So we know we aren't getting effective or 100% removal of DNA. Since this original transfection was a cDNA we cannot span an intron to solve the DNA problem. I feel like there is something simple we are missing, but we can't seem to figure it out. Any ideas would be greatly appreciative.
are you absolutely certain that it's not contamination or a problem with primer-dimers?
don't mean to be critical, just that those are easy fixes
what happens when you do everything exactly the same but leave out the DNAse step?
what do you mean by 'same template'? you are just running your RNA (prior to the RT step) through a straight-PCR, right?
I don't think they are dimers. Are band size is 320 bp. That is the size of the band we are seeing.
We haven't tried leaving out the DNAse step. What is your thinking? Wouldn't that just give us more DNA to start with?
Yes, by same template we are running the RNA sample straight through regular PCR. When we do this we see the same 320 bp band.
Also our Hela wild type cells don't express this gene. I don't know if this is important or not.
oh, hey, yeah, if your Hela cells don't normally express it, that's way important...do you have any RNA from un-transfected cells as a control? do you see the band in either PCR method when you use virgin RNA? this would be the absolute rule-out for contamination
As far as the DNAse step, I have a hunch and I was just curious if you had tried it w/out, and what you had seen...sorry don't mean to be cagey but I don't really want to go into detail cuz my theory's pretty rough. but anyhow, if there's a bunch of DNA in there still, then your DNAse isn't working so great...perhaps get some amp-grade DNAse from another source and try a separate treatment?
I don't think we have any RNA collected from untransfected cells, but we could grow a batch and test easy enough.
What about using the poly a tail of the rna? what is this all about?
in reference to what? what are you asking?
when I amplify my cDNA is use an oligo (dT) primer...this should give a higher prevalence of mRNA that gets turned to cDNA, vs other RNA species in the RNA prep; it is based on the polyA tail...is that what you mean?
I wonder if I could make use of the poly a tail on the mRNA to generate a cDNA that would be different than my cloned cDNA. From there I could PCR with a forward gene specific primer and a reverse poly A primer.
Would I use something like an oligo dt primer to generate the cDNA? what are the differences between oligo dt and other methods like hexamers?
If I use an oligo dt I see many companies offer them pre-made, but in different lengths. What length should I use and why?
I make my cDNA single-stranded (no forward and reverse; just one primer) some people perform a second-strand synthesis reaction; it's not really necessary...ssDNA will work just fine in your realtime and I think that's mostly about preference
oligo dT's aren't random like hexamers. They consist of strings of A's, which is how they are selective for mRNA. random hexamers just sort of allow the polymerase a place to start DNA synthesis from any old nucleic acid sequence
as far as different lengths and stuff, everyone has a preference...I use oligo (dT)VN 23 from NEB; it's not the cheapest one out there but it works well for me and the theory behind it is good. here is the link
but again, it's all about preferences and there are many ways to do it.
thanks for the tip. I get my primers from IDT, they said pretty much the same thing. I ordered an "anchored dt oligo" from them. I guess I'll give that a shot
Thanks for the tips