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I am doing relative quantification to compare gene expression between a control with several mutant backgrounds using qPCR.

Looking online I find several different sources suggesting different values that indicate high, low, and no gene expression. Some say that Ct > 35 = no expression, others say > 40 = no expression.

Where do you draw the line with Ct values and why?

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    $\begingroup$ I find that reaction chemistry often determines the upper range of usable Ct values. With intercalating dyes (SYBR), Ct values >~32 can start to blend with NTCs, but a melt analysis can help you tell if it's a true signal or not. With fluorescent probes, I generally count any Ct with decent technical reps, but in principle, a well designed reaction with good amplification efficiency should have single-copy Ct values at or below ~38 cycles, making quantitative interpretation of higher Ct values dicey (especially if you haven't established the linear range of the assay). $\endgroup$
    – MikeyC
    Apr 1 at 17:11

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Can't you just do a negative control and use that to calibrate?

Alternately, run the DNA from your PCR on a gel with a positive control indicating expression. Literally zero expression should mean that the correct band never comes up even after 40 cycles or whatever. And if you do see a band even in a negative control lane, that means that you have contamination and you should probably do something about that!

I think it's also reasonable to use those thresholds that you mention.

Overall, what I'd say is that PCR (and particularly Ct type values) are finicky and while the basic amplification is robust, there are tons of factors that affect the kinetics of your amplification curves. That's why it's so important to run controls in every qPCR run- you are correcting for all the weird batch effects of your reagents, primers, etc. in your master mix.

Every primer set will have slightly different kinetics, and so will every enzyme, so if you want to be really rigorous you would establish a threshold for every possible methodological permutation and reaction setup. However, that's a lot of work. I'd rather just run the final product on a gel and say yup or nope- it should be pretty obvious.

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    $\begingroup$ Thanks for your response. You're right, I definitely should have used a negative control. Unfortunately its too late now (I was doing an undergrad project and my time in the lab is up so i'm just left with the data). $\endgroup$
    – aquaporin
    Mar 31 at 19:08

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