How big of a relative fold change in RNA expression can I reasonably expect to be able to quantitate using qRT-PCR? I have a highly expressed gene that I'm knocking down, and I know (via a reporter assay) that I'm getting *at least* a 400-fold knockdown. But I'm hitting the signal-to-noise barrier with my reporter assay, so it may actually be much higher than a 400-fold knockdown. If I switch to qRT-PCR to quantify RNA levels, is it possible to accurately measure above a 400-fold change in expression? Is there a upper limit to a fold change I might be able to measure?
qPCR is one of the techniques with the highest dynamic range, so you can probably determine differences in expression much higher than 400 fold. Since I don´t know how familiar you are with qPCR, I´ll start with a brief primer: the more of your target in the sample, the lower its Ct will be. If you diminish the amount of target in half, the Ct increases in one unit. That is, if your original sample has a Ct of 23 and then you dilute the sample in half and run another qPCR, you´d expect its Ct to be 24. If you were to dilute your original sample 4 times, then its Ct would increase to 25. Now, to answer your question, since I wouldn´t trust a Ct over 35 for quantitation (Cts higher than 35 could be due to contaminant DNA), the answer depends on the Ct of your gene expressed in your basal conditions. If your highly expressed gene has a Ct of say 15 before knocking it down, and after knocking it down its Ct reaches 35, (but the Ct of your endogenous control doesn´t change*), then the difference in expression is 2e20, which is about a 1,000,000 fold. So yeah, you could easily measure a 1 million fold change with quite some certainty. It all comes to the Ct of your gene in basal conditions.
* In order to be sure that the difference in Ct is really due to differences in expression levels and not due to you adding more or less sample to your PCR reaction, you need to simultaneously quantify the expression of an endogenous gene which won´t be affected by your treatment, so you can correct for these changes when doing your calculations. For more information, read about the Delta-Delta Ct method for relative quantification.
Best Regards,
Alfonso
I agree Alfonso you can detect this much of fold change and you should add a reference gene (a housekeeping gene)
I'll add another method. If the exact change (400000) is important in your design you can try relative expression via standard curve method.
It's well detailed in the link on page 34
https://www.gu.se/digitalAssets/1125/1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf
In a typical qPCR the PCR efficiencies are taken as 2, but if the amount of mRNA is very low in the sample this efficiency is expected to change. The method I recommend gives you the exact PCR efficiency of the specific primer in the specific RNA sample. It uses too much RNA but if you have just 2 samples (knock-down and WT) it would not be a problem.
Best
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA