I am using the Qiagen precursor miRNA assay to detect my miRNA. As instructed, I treated the RNA with DNase I and set up a No RNA control and a No RT control. After RT and qPCR, I always got about the same Ct value in the No RT control with those of my samples (The no RNA sample was fine, showing a N/A). But, in the internal control, which I used the Human U5 RNA provided by Qiagen. The No RT control showed a N/A or a much lower value in terms of Ct.
After several trys, I suspected that my DNase I did not work well. I bought a shrimp nuclease that supposedly targets the dsDNA only and included it in the RT step, followed by a 65 oC deactivation. After proceeding to the qPCR, I still got the similar Ct value in all my samples including the No RT.
I can not figure it out. Anybody with experience in this kind of problem, I would appreciate your input. Thanks!
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Weird No RT control problem in the precursor miRNA experiment
Started by cathywill, Jan 28 2010 11:45 AM
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