Quantitative PCR: Theoretical Considerations with Practical Implications

Raeymaekers, Luc

doi:10.1006/abio.1993.1542

Cited by 224 publications

(152 citation statements)

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“…higher the concentration of the internal standard, the more likely the primers will bind and amplify it rather than the target. Gel electrophoresis may therefore be performed and a comparison can be made of ethidium bromide stained standard and target amplicons to allow target quantification 104 . There is still however, approximately 10% error and this method may still not result in absolute quantification as differences in amplification efficiency between target and competitor will remain undetected.…”

Section: Pcr Reaction and Conditionsmentioning

confidence: 99%

The GH–IGF-I axis and breast cancer

Laban

Bustin

Jenkins

2003

Trends in Endocrinology & Metabolism

109

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Section: Pcr Reaction and Conditionsmentioning

confidence: 99%

The GH–IGF-I axis and breast cancer

Laban

Bustin

Jenkins

2003

Trends in Endocrinology & Metabolism

109

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“…The ratio of the signal generated from the defined amount of heterologous template to target cDNA was quantified using the Image Processing program Win Cam 2.1. To validate the method of quantitation, measurements of template dilutions (standard cDNA and target mRNA) were carried out basically as suggested by Raeymaekers [15]. It is possible that a factor present in particular mRNA preparations (i.e.…”

Section: Reverse Transcription and Quantitative Polymerase Chain Reacmentioning

confidence: 99%

Regulation of ob gene mRNA levels in cultured adipocytes

1996

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mRNA levels of the ob gene product, leptin, were investigated by quantitative competitive RT-PCR in a mouse cell line (3T3-L1) which can be induced to differentiate into adipocytes. During conversion to fat cells, the level of leptin mRNA increased several-fold and in parallel to that for typical adipocyte markers like lipoprotein lipase, adipsin and glycerophosphate dehydrogenase. Leptin transcription, however, did not correlate with the size of the adipocytes measured as total triglycerides. On the other hand, mRNA levels for leptin in fully differentiated adipocytes were increased 2-3 fold by insulin. In contrast, free fatty acids exerted a concentration-dependent inhibition of leptin transcription while the corticosteroid dexamethasone and an elevation of intracellular cAMP displayed only marginal inhibitory effects on leptin mRNA levels.

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“…Also, competitive PCR determines the starting concentration of a target sequence by comparison to product accumulation in several dilutions of the reference. This practice is only valid if the amplification efficiencies of target and reference are identical and remain constant (6). However, the external standard in competitive PCR has frequently been observed to have an amplification advantage over the target and thus to be amplified more efficiently (9).…”

Section: Discussionmentioning

confidence: 99%