Synthesis of cDNA from mRNA is the first step of RT-PCR gene expression quantification. The quality of the cDNA is directly related to the quality of the RNA template. If possible, determine the RNA concentration of samples before using them in an RT reaction. Again, there are many methods for cDNA synthesis. Different RT enzyme kits are available (MultyScribe from ABI, SuperScript II from Invitrogen, AMVRT from Promega, Expand RT from Roche, and many others) using initiation with either oligo-dT or random hexamer priming. Random hexamers are preferred for analysis of non poly-adenylated RNA. Once prepared, first-strand cDNA should not be thawed and refrozen more than once. During PCR set-up, it has to be kept on ice and used immediately.
Once the cDNA has been synthesized, it remains to be verified that no DNA contaminates the samples. As the assay to determine mRNA quantity is based on PCR, the assay to detect genomic DNA contamination also needs to be performed using PCR (an agarose gel would not be sensitive enough). A PCR across an exon-exon boundary for an abundant mRNA should be used. A genomic DNA target may be used as control for the size of a potential genomic DNA contamination band. The primers ATG GCA AAT TCC ATG GCA and TGA TGA TCT TGA GGC TGT TGT C for the GAPDH gene generate an amplicon of 285 bp from cDNA and an amplicon of 453 bp from genomic DNA.
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