Where is absolute quantification qPCR used?

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Where is absolute quantification qPCR used?

Postby talkingtree » Sep 03 2018 2:39 am

Hi,

Majority of researchers use the relative method via reference gene. Does anyone know where absolute quantification qPCR is typically used? (and not digital PCR) Wondering why would people use this type of technique
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Re: Where is absolute quantification qPCR used?

Postby 29yrsExperience » Sep 04 2018 6:03 pm

Measuring gene expression is but one of many uses for qPCR. In this relative method, the number of copies of an mRNA (after they have been converted to cDNA) is normalized to the number of copies of mRNA of a reference gene. But sometimes you want to know how many copies of a genomic gene target are present in a sample- for example, maybe you want to know how many bacteria of a certain species (especially the unculturable ones) can be detected in an environmental sample, perhaps with samples taken in many different locations. In this case you are not interrogating message RNA, and you have no “reference gene.” So you use a standard curve of known quantities of the gene target. Digital PCR is a refinement of this method that substitutes probability algorithms and a statistically significant number of sampling iterations for the standard curve.
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Re: Where is absolute quantification qPCR used?

Postby talkingtree » Sep 05 2018 12:38 am

29yrsExperience wrote:Measuring gene expression is but one of many uses for qPCR. In this relative method, the number of copies of an mRNA (after they have been converted to cDNA) is normalized to the number of copies of mRNA of a reference gene. But sometimes you want to know how many copies of a genomic gene target are present in a sample- for example, maybe you want to know how many bacteria of a certain species (especially the unculturable ones) can be detected in an environmental sample, perhaps with samples taken in many different locations. In this case you are not interrogating message RNA, and you have no “reference gene.” So you use a standard curve of known quantities of the gene target. Digital PCR is a refinement of this method that substitutes probability algorithms and a statistically significant number of sampling iterations for the standard curve.


Ok that makes sense. Thanks for the input.
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