The qRT-rtPCR Control You Should Be Doing, But Probably Aren’t

Every man, woman, and dog is doing quantitative real time reverse transcriptase PCR (qRT-rtPCR) these days. It’s a great method to measure your favorite transcript’s expression levels. One of the big plusses (like the Swiss flag!) of quantitative PCR in general is its high sensitivity. 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.

Using DNase

People are super careful to remove contaminating DNA from their RNA. Careful RNA isolation and treatment with DNAse can persuade most people that their cDNA is free of genomic DNA contamination.

But DNAse treatment is scary. 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. You get results, but who knows what kind of artefacts are influencing their results?

The Control You Need for qRT-rtPCR

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. This will give you an RT sample and a negative RT control (–RT). Then, if you’re worried that genomic DNA might be messing up your results, run both these samples through the qPCR machine. 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.

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But 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. You’ve got a difference of 12 Ct values. That’s a difference of 2¹²-fold, which is 4096-fold! In other words, you’ve got 4000 times more cDNA than genomic DNA for your gene of interest. The genomic DNA contamination is so small, that it is not going to have any effect on your results.

On the other hand, if you have a lowly expressed gene that has a Ct of 30 and your contaminating DNA has a Ct of 32 again, then you’ve only got 2^2 which is only a 4-fold difference (I should have been able to do that math in my head but still reached for the calculator…). That might mess up your results.

Do You Always Need the Control?

I’m not going to give you any hard and fast rules here, just take it on a case-by-case basis. If you’re selecting for mRNA and you’ve got highly expressed genes, this control probably isn’t necessary. However, if your genes are expressed at lower levels , and you have no poly-A enrichment step in your protocol (if you’re studying bacterial or eukaryotic organelle genes, for example), you might want to seriously consider including a –RT control for every qRT-rtPCR reaction you run.

There’s my nugget of wisdom I learned doing a lot of qRT-rtPCR on weird genes. Take it or leave it, but keep in mind that you really don’t want to just quantify the cleanliness of your RNA sample!