Bizarre Contamination in PCR - (Jul/08/2009 )
Hi guys,
When ive been running gels I used get contamination in my no RT controls, but only in my experimental primers and not for the ones in my housekeeping gene. I then realized that my housekeeping gene primers did span an intron while those for my experimental ones didn't. So i reordered a set of primers that did span an intron, but I am still getting the same problem, with contamination at the same BP length in my RT controls. This is bizarre because, i would expect that by including an intron the band should be located higher up in my gel. So im stuck thinking that it must not be genomic contamination, but rather somehow the RNA that is in my no-RT controls is getting reverse-transcribed even in the absence of added RT-ase. But then this still wouldn't explain why I'm not getting any contamination in my other lanes, because if there is some RT-ase-like activity coming from my other materials, then it should be affecting them all with equal affinity. This has been driving me nuts. Any help would be much appreciated.
Thanks
mastcells on Jul 8 2009, 09:24 AM said:
When ive been running gels I used get contamination in my no RT controls, but only in my experimental primers and not for the ones in my housekeeping gene. I then realized that my housekeeping gene primers did span an intron while those for my experimental ones didn't. So i reordered a set of primers that did span an intron, but I am still getting the same problem, with contamination at the same BP length in my RT controls. This is bizarre because, i would expect that by including an intron the band should be located higher up in my gel. So im stuck thinking that it must not be genomic contamination, but rather somehow the RNA that is in my no-RT controls is getting reverse-transcribed even in the absence of added RT-ase. But then this still wouldn't explain why I'm not getting any contamination in my other lanes, because if there is some RT-ase-like activity coming from my other materials, then it should be affecting them all with equal affinity. This has been driving me nuts. Any help would be much appreciated.
Thanks
Think of any other source of your gene sequence contaminating any of your reagents. The most likely one is a plasmid containing your gene. In any case, DNase treatment of your RNA samples - prior to First strand synthesis, should take care of your problem.
mastcells on Jul 8 2009, 09:24 AM said:
When ive been running gels I used get contamination in my no RT controls, but only in my experimental primers and not for the ones in my housekeeping gene. I then realized that my housekeeping gene primers did span an intron while those for my experimental ones didn't. So i reordered a set of primers that did span an intron, but I am still getting the same problem, with contamination at the same BP length in my RT controls. This is bizarre because, i would expect that by including an intron the band should be located higher up in my gel. So im stuck thinking that it must not be genomic contamination, but rather somehow the RNA that is in my no-RT controls is getting reverse-transcribed even in the absence of added RT-ase. But then this still wouldn't explain why I'm not getting any contamination in my other lanes, because if there is some RT-ase-like activity coming from my other materials, then it should be affecting them all with equal affinity. This has been driving me nuts. Any help would be much appreciated.
Thanks
Agree with DNase treatment, since without RT it must be original DNA amplifying. Also, how many cycles are you running? I've found that even with DNase treatment of the RNA, it only takes a single molecule of surviving template to amplify, and after ~30 cycles I was seeing background bands of the correct size in everything. I think filter tips will help avoid DNA contamination from your pipettors, which I'm convinced is a major source, in case you're not using them already. There are also DNA decontamination sprays out there.
Sorry I didn't include that earlier, DNase is standard treatment, and when I've run into this kind of problem I've often gone back and done it a second or even third time. Aerosol filter pipet tips are standard as well. Cycle length is often 36-40 cycles. But I still dont understand how if it is genomic contamination that is surviving the DNase, why is it still showing up as the same length, when my primers span an intron? Shouldn't they be a larger fragment size? I use the UCSC Blat feature to take my amplified region from my primers and compare it to the genomic sequence. Im assuming that the black chunk of sequence inserted between my blue amplified sequence, that indicates an intron in the genomic sequence. Am i correct in assuming this?
mastcells on Jul 8 2009, 05:20 PM said:
Oh I'm sorry, I don't know what the UCSC Blat feature is.... Maybe I should, for I am always angry with people writing about stuff they don't know of (who was this funny guy answering the TOP10 problem?). Well, anyway, I had a similar problem with intron-spanning primers. It turned out to be pseudogenes, that had integrated into the genome somewhere else but lost their introns (lots of housekeeping genes do that!). Have you checked if your primer might amplify pseudogenes? Although, if you DNase them... should be no problem.
Sorry,
Minna
(typo)
It's still not clear to me exactly what you're doing, but you can also have RNA contamination in your cDNA, assuming you're doing this two-step. I treated my cDNA with RNAse H (not inhibited by RNAse inhibitors that may be in your RT reaction) before running the final PCR amplification of the cDNA. 36-40 cycles is a *lot*, do you really need to go that high?
While RNA contamination in my cDNA is something I've never thought of, I still can't understand how this is showing up only in one specific lane consistently.
To clear things up, this is what I've been doing/ testing for:
extracting RNA from mast cells
DNase'ing RNA
RT (include no RT-ase controls)
PCR using primers for my housekeeping gene H-RPL, and the gene I'm investigating, leptin (so far I have 4 samples: positive-RT leptin, Positive-RT H-RPL, No-RTase leptin, No RTase H-RPL).
Run gels
My problem is that I'm consistently obtaining contamination in my (no-RTase )leptin lanes but not in my (no RT-ase) RPL lanes. Both primers should be spanning introns (see my last post). Therefore I can't seem to explain why the contamination that does show up in my no-RTase samples is the same length as the predicted amplified region (should be longer because it spans introns) and also why my other lanes don't show any contamination. People have said that my PCR cycles are high, but that doesn't explain why I have never gotten any contamination in my RPL lanes, while getting contamination every single time in my leptin lanes, because each PCR sample is being made from the same batch of RT. Therefore if there is a tiny bit of genomic contamination that is being amplified to a great extent because of the large number of cycles, that large amount of genomic contamination should show up in all my lanes. And I can't call it a fluke, that by luck of the draw one sample may have received a bit more genomic contamination before PCR and thus ended up with significantly more after amplification, because I've repeated this maybe 10 times and I always get the same result.
Any advice would be greatly appreciated
bump