Primers amplify genomic DNA but not cDNA - (Aug/01/2012 )
I'm trying to look at the pre-spliced forms of two different genes (the wildtype and the replacement gene) in tissue that our collobrators sent from knockout mice. I can see the two different size bands when I test the primers with genomic DNA, but when I go to use cDNA (made from 2-3ug DNAse treated total RNA), I see nothing other than faint bands well below 100 (both bands should be between 850-1000bp). I know the problem isn't the cDNA, because when I use other primers I get a specific band of the right size for that particular gene, but I also know the problem shouldn't be the primers as they work with the genomic DNA.
Any thoughts anyone, pretty soon I'm going to be banging my head against the desk in pure frustration as I'm running out of options to try.
Are you sure you're getting pre-spliced forms? That usually require nuclear fraction RNA. How do you isolate RNA?
I'm affraid from "total RNA" kits you won't get unspliced RNA in sufficient amounts, if that was true, people would get get two types of bands in their intron spanning PCR assays even when having no DNA contamination.
Your first primer, it's probably in intronic region, right? Are the other primers in introns too?
The forward primers are actually in the exon (about 200 bases from the end) and the common reverse primer is in the intron. I homogenize the tissue in trizol and follow the manufactuers instructions for RNA isolation. I really hope I won't have to do a nuclear fractionation to get the unspliced RNA.