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!!