Limitations Imposed by Heteroduplex Formation on Quantitative RT–PCR

Henley, William N.; Schuebel, Kornel E.; Nielsen, David A.

doi:10.1006/bbrc.1996.1319

Cited by 34 publications

(14 citation statements)

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“…(ii) Competitor RNA must be transcribed in vitro, which adds an additional enzymatic step; furthermore, once transcribed it may degrade during long-term storage. (iii) Heteroduplex formation between the nearly identical standard and target can result in variable sensitivity and accuracy (Henley et al 1996); use of DNA competitors with primer binding sites flanking unrelated internal sequences (mimics) (Siebert & Larrick 1993) eliminates that problem, but causes new ones. (iv) The quantification of competitor fragments generated by restriction digestion requires the dilution of PCR products before digestion, introducing one source of error; a second source of error is the restriction digest itself, as it may not completely digest the competitor.…”

Section: Rt-pcr Quantification Strategiesmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,440

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Section: Rt-pcr Quantification Strategiesmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,440

2,448

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show abstract

“…The fact that the accuracy of the new method is comparable with that of the RTDN PCR method, where heteroduplex formation between the target and the mimic sequences (20) does not interfere with the accuracy, suggests that heteroduplex formation does not present a problem in our system. If heteroduplexes occurred, they would have a loop of 96 nt (Fig.…”

Section: Generation Of Rna Mimicsmentioning

confidence: 83%

A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis

Troubetzkoy²,

Nguyen³

et al. 2000

Nucleic Acids Research

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“…This statement is based on practical issues, such as the precision of den- sitometric determinations of gel bands (1,17) and compensation of heteroduplex formation when homologous competitors are used (28). It should be noted, however, that there is not any theoretical background to choose the point of equivalence for relative quantifications.…”

Section: Fig 2 Effect Of the Initial Target Amount (T 0 ) On The Camentioning

confidence: 99%

Bias in Estimations of DNA Content by Competitive Polymerase Chain Reaction

Alvarez

Depino

Podhajcer

et al. 2000

Analytical Biochemistry

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Limitations Imposed by Heteroduplex Formation on Quantitative RT–PCR

Cited by 34 publications

References 0 publications

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis

Bias in Estimations of DNA Content by Competitive Polymerase Chain Reaction

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