I am trying to generate cDNA from MCF-7 cells. So far, I have been able to extract total RNA using a combination of Trizol and Qiagen RNEasy kit. My A260/280 values are all above 2.0 and the quality of RNA seems to be good. I then proceeded to use the EcoDry Random Hexamers kit for generating my cDNA.
Is DNAse I treatment absolutely necessary before cDNA preparation? In the case of OligodT it is understood that only mRNA will be amplified (so I gather DNAse1 treatment can be skipped), but the random hexamers kit has me stumped. I will be grateful for your insights.
Hi Subramaniyam,
I don't know about Qiagen RNEasy kit, but all RNA extraction kits i've used so far (Promega, Zymo Research and Macherey-Nagel) include a DNAse treatment in their protocol. You therefore shouldn't worry if you performed the DNAse step while extracting RNA. If you didn't, a DNAse treatment would be advisable. How will you be sure that the PCR product was amplified from the cDNA or left over DNA?
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