using a human plasmid and a mouse sample for qRT-PCR

[ambarrie]

I am currently trying to perform qRT-PCR to examine mouse sk3 mRNA. However, to create the standard curve, I do not have mouse sk3 plasmids, only human sk3 plasmids. I have aligned the human sk3 and mouse sk3 sequence using BLAST2, and I have found a primer that exactly matches both sequences. However, the amplicon that I am creating (~100 bp) differs between mouse and human by 2 base pairs. These base pairs are close to the 3' end of the reverse primer. Does anyone have any guidelines or suggestions for such an experiment? I can't seem to find anything on pubmed or google about it, and I was wondering if a 2 bp difference in the amplicon that close to my reverse primer would be all right. Thanks so much!!

[danfive]

This may may work out for you. If the two bases are not in your primer sequences, don't worry about them. Just keep in mind that if the bases are GC, CC, GG and are closer to other G or C repeats you may need to melt them with a higher temp.