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Gene expression in colonic tissues using qRT-PCR

RNA Housekeeping gene colon tissues

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#1 Shima Bibi

Shima Bibi

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Posted 20 September 2016 - 07:23 PM

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?



#2 bob1

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Posted 20 September 2016 - 08:54 PM

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.







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