quantitate wild type and mutant mRNA - (Apr/18/2011 )
I have transfected a cell line with an untagged mutant form of a gene. When trying to determine how much of the mutant gene is being expressed, I first attempted to use a western blot, but with the mutant being untagged, was not able to distinguish between the exogenous mutant and the endogenous wild type version of the gene.
One thing I would like to do is to use qPCR (or another technique if possible) to quantitate the amount of wild type AND mutant mRNA in the cells. I'm assuming the best way to do this is to design primers encompassing the mutant region, and running a qPCR reaction for the 2. I have minimal experience using qPCR - I've only run a few experiments (really by following someone else around using premade primer assays from Applied Biosystems).
Does my approach to this seem legitimate? Is there something I am missing, or something I should think about before going through with the primer design and ordering? Thanks in advance.
For quantitation to distinguish between wt and mutant, there has to be a region where they differ in sequence (you should align them in clustalW or similar to see the differences). Then you design primers in that region (at least 2 bp of 3' missmatch on one primer), you test for specifity and then you can do the qPCR.
Can you post the sequences (link or attachment)? I may be able to tell you if it's possible.