RNA isolation from full thickness colon - RNA isolation, colon (Dec/16/2008 )
I have been trying to isolate RNA from colon tissue (Qiagen Midi Kit) for almost 2 years now. The goal is to obtain cDNA via RT-PCR. Either I don't get any product or I get contamination of the product in my negative controls. I have thrown everything away taken every precaution (keeping controls and samples apart, keeping tubes closed when not in use, thouroughly cleaning my pipettes with 1% bleach).
One thought was that inefficient RNA isolation leads to DNA contamination that is showing up in my controls. The only control that supports that is my Minus RT (with RNA) that showed up. The other two (minus RNA and minus cDNA for PCR) also show up and I have no explanation for that unless there is just a ton of DNA around from the isolation that contaminates everything. I do everything in the same space (different days and thorough cleanings at the end of the day and before beginning a new experiment). Our lab is small, so having a different space is not an option. I use filtered tips, I wear a different lab coat for RNA isolation and RT-PCR, and change gloves frequently when both procedures. Also, I am the only one having trouble with this. The other people in the lab work with liver IAmbion mini kit) and breast tissue (RNeasy Lipid Kit) and they are not having this problem.
If the RNA isolation is part of the problem in that it is not removing all of the DNA, does anyone have any suggestions for how to solve this? I use the Qiagen Midi Kit on full thickness colon tissue (we were initially under the impression that we were only getting colon mucosa but it turned out not to be the case.)
I am ready to quit grad school !!! I hope this is the right section for this topic. Thanks for reading and any suggestions will be much appreciated.
I have you tried using separate pipettors for the before and after experiment so that when you set up the experiment your pipettors have never been exposed to your PCR product? This has helped me in the past. You might want to look up the information on cleaning your pipettors too - I think that some of the parts can be autoclaved. You could also buy some stuff called DNA away that decontaminates surfaces.
You could bet that almost all RNA extraction methods have DNA contamination. Use DNaseI treatment and design RNA specific primers are the way to go. All those precautions of changing lab coat, gloves, using DNA decontamination solutions, using designated bench and pipettes etc. are just silly and being over-emphasized