Gene expression in colonic tissues using qRT-PCR - (Sep/20/2016 )

HI,

I have extracted RNA from colonic tissues of mice (dextran sodium sulphate (DSS) create mice) with trizole. Then I have used the Dnase one to digest the DNA and then cleaned up using the RNAasy column. Finally adjusted the concentration to 200ug and then made cDNA. When I ran the qRT-PCR for reference genes (Actin, 18s and tubulin) the Ct values are 2 cycles different among the treated and untreated group. DSS mice colonic tissues have less expression of the reference gene, and for the target gens there is no expression because I am getting Ct 36. From where to start the trouble shooting?

That's the whole point of having reference genes; you can compare expression levels relative to the reference genes. the most common method for doing this is the delta-delta ct method of Pfaffl.