The Inherent Quantitative Capacity of the Reverse Transcription-Polymerase Chain Reaction
| Autor: | Daniel J.J. Carr, Viviana C. Falco, Bryan M. Gebhardt, William P. Halford |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Coefficient of variation
Biophysics Herpesvirus 1 Human Biology medicine.disease_cause Biochemistry Immediate-Early Proteins chemistry.chemical_compound Primer dimer Chlorocebus aethiops medicine Animals RNA Messenger Vero Cells Molecular Biology DNA Primers Reverse Transcriptase Polymerase Chain Reaction Cell Biology Molecular biology Reverse transcription polymerase chain reaction Standard curve Herpes simplex virus chemistry Linear range Primer (molecular biology) DNA |
| Zdroj: | Analytical Biochemistry. 266:181-191 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1998.2913 |
| Popis: | The quantitative capacity of the reverse transcription-polymerase chain reaction (RT-PCR) is generally underestimated. In this study, PCR and RT-PCR products were amplified from serially diluted DNA and RNA templates, respectively, using a 35-cycle PCR. In the approximate 30- to 100-fold range of template input above the lower limit of detection, herpes simplex virus ICP27 RT-PCR product yield was dependent on the logarithm of template mRNA input (r2= 0.99). Likewise, regression analysis indicated that yields of interleukin-12 p40, herpes simplex virus DNA polymerase, and interferon-γ PCR products were dependent on the logarithm of template DNA input over 40- (r2= 0.98), 60- (r2= 0.96), and 100-fold (r2= 0.99) ranges, respectively. This quantitative relationship appears to derive from the competition for reactants between specific PCR products and nonspecific primer–dimers that occurs at limiting concentrations of template. Although primer–dimers are not generally considered a common feature of PCR, 30 of 32 primer pairs tested in this study produced primer–dimer amplification in the absence of template. Because the coefficient of variation in replicate PCRs was typically 10–20% in the linear range, the precision of PCR was sufficient to measure 4-fold differences in template concentration. Thus, with statistically adequate sample numbers, an appropriate standard curve, and the inherent quantitative capacity of the method, differences in the abundance of a mRNA species are measurable by 35-cycle RT-PCR. |
| Databáze: | OpenAIRE |
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