QPCR

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hotdog
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QPCR

Hello , Does anybody with experience in QPCR have any idea about whether standard concentrations must be exactly known before using it for quantitative PCR. If we are doing relative quantification with houskeeping gene is it necessary to know exact concentrations of standards ?is it necessary to have sample dilutions exactly should lie with in the standard dilution range ? is extrapolation of the sample concentration from standards concentration is unreliable?
Thanks a bunch in advance
hotdog

smartee
smartee's picture
hotdog wrote:Hello , Does

hotdog wrote:

Hello , Does anybody with experience in QPCR have any idea about whether standard concentrations must be exactly known before using it for quantitative PCR. If we are doing relative quantification with houskeeping gene is it necessary to know exact concentrations of standards ?is it necessary to have sample dilutions exactly should lie with in the standard dilution range ? is extrapolation of the sample concentration from standards concentration is unreliable?
Thanks a bunch in advance
hotdog

No, and I prefer not to run standard curves with the samples because it's just too easy to cross-contaminate. And it's a waste of the reagents as well.

You can use the delta-delta-Ct method as long as the efficiencies of the assays for your genes of interest and the housekeeping gene(s) are the same. ABI web site has a rather confusing paper on this method, so I'd suggest that you get in touch with tech support of either your equipment or reagents. Our lab has a Stratagene machine and their people been pretty helpful in helping figure it out.

Sergi Castellvi-Bel
Sergi Castellvi-Bel's picture
Hi there,

Hi there,

If you doing absolute quantification and you want to get your result in concentration units, you should know indeed. In case you're performing relative quantification and you want to check your assay slope and performance, different volumes or dilutions without a absolute value should work also.

Best regards

Adolfo_Ferrando
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I am sorry smartee, but if

I am sorry smartee, but if you are to use the Delta Delta Ct method you must check the kinetics of your reaction and a standard curve is the best way to assess it. There are mathematical methods to correct for not "perfect" kinetics but I prefer to get true dta than doing any mathematical fitiing that I do not fully understand.
Adolfo

jetobin
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For relative quantification,

For relative quantification, it is not necessary to know the exact concentrations of the standards. As long as the dilution factor (ie, 1:10 or 1:2) between points in your standard curve is accurate, you are ok. Your standard curve should be over at least 2 orders of magnitude so that extrapolation to points beyond the range of the curve will be more accurate.

smartee
smartee's picture
Adolfo_Ferrando wrote:I am

Adolfo_Ferrando wrote:

I am sorry smartee, but if you are to use the Delta Delta Ct method you must check the kinetics of your reaction and a standard curve is the best way to assess it. There are mathematical methods to correct for not "perfect" kinetics but I prefer to get true dta than doing any mathematical fitiing that I do not fully understand.
Adolfo

Yes, of course, but the kinetics of the reaction can be checked when designing the assay, and as pointed out by jetobin, there is no need to know concentrations. I usually find a tissue with high expression levels of a particular gene of interest, and optimize my assays to ~100% efficiency in 10x dilutions of it's cDNA. Once the I know that a paticular set of primers and/or probes works consistently, I can use it without running the standard dilutions every time.