Effect of collagenase on qpcr results - (Mar/25/2011 )
I recently started performing qPCR to look at gene expression in fibroblasts that are embedded in collagen matrix after various conditions. Initially, to remove the cells from the gel, I tried using collagenase. However, I read in a publication that using collagenase skews the expression results and so its not ideal. So, I used a purely mechanical way of breaking down the gel & using lysis buffer to help extract the mrna as described in the publication.
However, with my results I notice that with the second method, I see high Ct values even for housekeeping gene (between 25-30) and my gene of interest values are beyond 40. Earlier, with the collagenase, I saw the housekeeping gene at around (10-15) and gene of interest at around (35-40..still iffy numbers to conclude anything).
So, my question is should I go back to using collagenase just because it gives me relatively better numbers? Anyone else use any other methods for mrna extraction when cells are in collagen matrix? I'm really frustrated!!
Sorry for the long post. Any help would be mucho appreciated!
Unfortunately I do not have experience extracting RNA from cells embedded in a collagen matrix. Nevertheless I can give you some hints about what to do to get around your low Ct problem:
1. Design new assays. Sometimes it is just a matter of getting the right primers to get significantly better Ct values. I've always designed at least three qPCR assays and ran them side by side. The one that worked the best was the one I chose to work with
2. Use more RNA in your cDNA synthesis, or be super careful and quick when extracting RNA. Some RNAs are very unstable and you may be dealing with one of those
3. Use a different housekeeping gene. If your gene of interest is showing up more than 10 PCR cycles after your housekeeping gene, then analyzing the expression of your gene of interest with that housekeeping gene is not really the most appropriate thing to do (their amplifications are too different to be considered similar enough for comparison).