![]() You’ll (hopefully) see a Ct value for your RT sample of, say, 20 (which means it takes 20 cycles of PCR for the fluorescence to exceed certain threshold), and a higher Ct value of, say, 32, for your –RT control.īut wait! You’ve got a Ct value of 32 for the –RT control! Shouldn’t you not have any Ct value at all?! Well, hang on just one sec, don’t freak out yet. Then, if you’re worried that genomic DNA might be messing up your results, run both these samples through the qPCR machine. This will give you an RT sample and a negative RT control (–RT). So, we finally get to the point that I’ve been leading you towards: the new control for your next qRT-rtPCR experiment! When you do your reverse transcription reaction, also do a reaction with no reverse transcriptase enzyme. You get results, but who knows what kind of artefacts are influencing their results? The Control You Need for qRT-rtPCR To get rid of your genomic DNA, you incubate your fragile RNA sample at warm temperatures for a reasonable length of time, with an enzyme that happens to have a bit of a taste for digesting RNA, too! You can over-digest your RNA sample, removing all DNA but also degrading your RNA to some extent. Careful RNA isolation and treatment with DNAse can persuade most people that their cDNA is free of genomic DNA contamination.īut DNAse treatment is scary. People are super careful to remove contaminating DNA from their RNA. ![]() In principle, it can detect and quantify one molecule of DNA! In reality, it’s not quite so perfect (but it gets close)! This sensitivity is also a major drawback, because any contaminating DNA is detected just as easily as the cDNA you lovingly reverse transcribed from your painstakingly purified RNA. ![]() One of the big plusses (like the Swiss flag!) of quantitative PCR in general is its high sensitivity. It’s a great method to measure your favorite transcript’s expression levels. Every man, woman, and dog is doing quantitative real time reverse transcriptase PCR (qRT-rtPCR) these days. ![]()
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