Over the thumb, real-time qPCR can quantify (with sufficient precision) when the ct-value is lower than 30. Given the amplification efficiency is close to 2 (100%), a log2FC of 1 unit will shift the ct by 1 cycle. Thus, when the ct of the control is at 23, a down-regulation by 2^(-7) will shift the ct to 30, where the target sequence can still be quantified. So the answer depends on the ct value under control conditions. The answer is "yes" when this ct is lower than (round about) 23.

PS: Sensitivity, as Korcan wrote, is not related to the problem. The important parameter is the dynamic range of the method, and this range is quite large for rtqPCR. One can get reliable quantifications for samples with ct from around 15 (some say even for 10) down to 30. These are at least 15 cycles, referring to a fold-difference of 2^(30-15) = 30000-fold (optimistic: 2^(30-10) = 1-million-fold). Give and take some bunsh of horse hairs.

qPCR is much more sensitive and reliable than microarray, with a well designed assay and correctly selected reference gene you will cover a wide range of up and down-regulations.

Over the thumb, real-time qPCR can quantify (with sufficient precision) when the ct-value is lower than 30. Given the amplification efficiency is close to 2 (100%), a log2FC of 1 unit will shift the ct by 1 cycle. Thus, when the ct of the control is at 23, a down-regulation by 2^(-7) will shift the ct to 30, where the target sequence can still be quantified. So the answer depends on the ct value under control conditions. The answer is "yes" when this ct is lower than (round about) 23.

PS: Sensitivity, as Korcan wrote, is not related to the problem. The important parameter is the dynamic range of the method, and this range is quite large for rtqPCR. One can get reliable quantifications for samples with ct from around 15 (some say even for 10) down to 30. These are at least 15 cycles, referring to a fold-difference of 2^(30-15) = 30000-fold (optimistic: 2^(30-10) = 1-million-fold). Give and take some bunsh of horse hairs.

As Korcan mentioned, qPCR is more sensitive than microarray but it is also more precise, especially for small differences. So with anything over 2-fold differences, you should have high confidence of the result given by qPCR, as long as you are in the dynamic range of your primers efficiency. Anything lower than 2-fold differences, you will need to have more replicates as qPCR loses its precision at this level.

Similarly, if your target appears after cycle 30, you will need more replicates again as precision of qPCR is low in this area (meaning it will be harder to find small differences in this region [29-34 Ct] than for a target appearing in region < 29Ct.

Good luck with your validation.

CL

Claude, i doubt that "qPCR [...] it is also more precise [than microarrays], especially for small differences.". To my experience, it is the other way around (and the precision has nothing to do with the size differences - small differences won't get clear when the precision is small, but the precision does not depend on the actual difference).

The technnical variance of qPCR is typically around 0.125 c², i.e. the SD is 0.25 cycles, what translates to an SD of 0.25 for the log2 concentrations and or 0.35 for the log2 fold-changes. This, in turn, translates roughly in a 95% prediction interval from 60% to 160%.

In microarrays, the SD of a log2 fold-change is typically 0.2, translating to an interval from 75% to 130%.

Your dependency on the ct is a problem of low analyte concentration, where the Poisson distribution limits the achieveable precision. This is independent of the methods. However, such low concentrations can't be detected with microarrays (at least not in special cases with sophisticated preamplification techniques).

Sure and even higher differences are measureable

you have 40ct values so theoretically =2^40

But validation of modern microarray output with qPCR is not necessary anymore

(or do you validate your qPCR with NothernBlottings?)

Vincent, I don't think that 40 cycles are possible, or if, only for lower efficiencies. A single molecule amplified with 100% efficiency will give a ct around 32-35 (depending a little on the chemisty, optics, and threshold setting). Thus, ct values > 35 do not represent any quantitative result (either there is or is not a copy in the PCR). Further, ct values close to 0 are also no reliable measures of the target concentration, mostly because the amplification curves won't have any recognizeable exponential phase and the background correction won't work (I think that these problems could be solved by a clever design and interpretation of the fluorescence readings... but what for? - and available machines and software clearly will give erroneous results for "too high concentrated" samples).

If you assume an amplificatiuon efficiency of 80%, the highest possible ct values (for detecting a single molecule) will raise to 40-44, but then the dynamic range would have to be calculated as 1.8^40, what is just 1.5% of 2^40...

Hi Jochen,

I don't agree, if have made a few primers pairs the last 10 years ( all deposited at www.RTprimerDB.org) , the cDNA calibration curve we used was diluted 2,4,8,16,32,64,128,256 times .

the delta CT in reaction was nearly 8, given an efficiency of almost 100% ( 2^8=256)

Vincent, I did not understand with what you disagree. My statement was that a dynamic range of 40 cycles is not possible if the efficiency is close 100%. I did not say that the efficiencies are typically much smaller. Th 80% was a made-up example to demonstrate how this would make such a dynamic range of ct-values possible (and that this would still not widen the dynamic range of concentrations!).

I made many primers, too, and never was the estimated efficiency based on dilution series below 95% (although I have seen assays from collegues where the efficiencies were closer to 90%, though - so it can happen). I know this well, and I always stress the point that a reliable quantification with "low-efficiency assays" is unlikely (if the efficiency is suboptimal, it may vary between samples and - even worst - may not be constant during the first cycles)

Hi Jochem,

You are correct a dynamic range of 40 cycles is not possible

but a thought the question of Navjyoti was if the qpcr could handle the 7 ct values in range,for validating his array i think this should be no problem at all.

So we both agree :)