To study gene expression with qPCR, I'm using 16s as a reference gene.
16s is of course very, very abundant so I need to dilute my cDNA a lot. However, I don't want to overdilute my cDNA as then my GOIs/target genes might be >30 Ct.
What's the lowest Ct that's still typically accurate?
I could of course dilute my cDNA for GOIs and for 16s separately, but that feels like it would add a layer of error that I don't want.
You're right, not ideal to use different dilutions for different genes, there is a limit to how many genes you can have in the 'ideal Ct range'. Do a standard curve of 16S (and your target gene while you're at it) at serial dilutions to see if high Ct values affect your PCR efficiency. If it is still linear at the higher concentration then you are good to go!
No problem, good question. I believe 100 +/- 10 % (yes the efficiency can go over 100 %) is the acceptable range by convention. The formula for efficiency (E) is E = -1+10(-1/slope). Therefore a slope between approximately -3.6 and -3.1 indicates acceptable efficiency.
Adrian Abdo
Thank you again. Although, wouldn't the number of other data points affect the calculated efficiency result a lot?
If I theoretically have a very large dilution series, even if 1 point is massively out of range, the other values will compensate it for it and give me a good efficiency value.
Does that matter?
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