Quantitative PCR reverse transcription

Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., Takeda, N., and Katayama, K. (2003). Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. /. Clin. Microbiol. 41, 1548-1557. 

Another important set of multiplexed assays monitor mRNA transcript levels. The expression level of all the genes involved in a known signal transduction pathway or other selective genes can be monitored simultaneously as a way of following compound effects on a cell. The current technologies for multiple mRNA detection include quantitative reverse transcriptional PCR (qRT-PCR), qNPA (quantitative nuclease protection assays), mass array assay technologies and branched DNA detection on Luminex beads (Panomics). The applications of such multiplexed in vitro and cell-based detection systems should provide more predicative information in hit finding and lead characterisation. 

Lowther, J. A., Avant, J. M., Gizynski, K., Rangdale, R. E., and Lees, D. N. (2010). Comparison between quantitative real-time reverse transcription PCR results for norovirus in oysters and self-reported gastroenteric illness in restaurant customers. /. Food Prot. 73, 305-311. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

Haberhausen, G., etal. (1998). Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays. J. Clin. Microbiol. 36,628-633. 

Because the 5-HT4 receptors mediate physiological effects in the heart, gut, and CNS (132), splice variants of this receptor are thought to be involved in atrial arrhythmia, irritable bowel syndrome, and neurodegenerative diseases. Medhurst et al. (132) used TaqMan real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. They... 

Martell M, Gomez J, Steban JI, Sauleda S, Quer J, Cabot B, Esteban R, Guardia J (1999) High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA. J Clin Microbiol 37 327-332... 

S. J. Wall and D. R. Edwards, Quantitative reverse transcription-polymerase chain reaction (RT-PCR) a comparison of primer-dropping, competitive, and real-time RT-PCRs. Anal Biochem 300(2) 269-273 (2002). 

Baehner FL, Maddala T, Alexander C, et ah A Kaiser-Perma-nente population-based study of ER and PR expression by the standard method, immunohistochemistry (IHC), compared to a new method, quantitative reverse transcription polymerase chain reaction (RT-PCR). ASCO Breast Cancer Symposium. 2007 Abstract 88. 

Cl. Castello, R., Estelles, A., Vazquez, C., et al.. Quantitative real-time reverse transcription-PCR assay for urokinase plasminogen activator, plasminogen activator inhibitor type 1, and tissue metalloproteinase inhibitor type 1 gene expressions in primary breast cancer. Clin. Chem. 48, 1288-1295 (2002). 

Cheung, I.Y., Cheung, N.-K.V. (2001) Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR. Clin Cancer Res, 7, 1698-1705. 

Lamar RT, Schoenike B, Vanden Wymelenberg A, Stewart P, Dietrich DM, Cullen D (1995) Quantitation of fungal mRNAs in complex substrates by-reverse transcription PCR and its application to Phanerochaete chrysosporium-coloTiized soil. Appl Environ Microbiol 61 21 —2126 Lashermes G, Houot S, Barriuso E (2010) Sorption and mineralization of organic pollutants during different stages of composting. Chemosphere 79 455-462... 

Cook L, Ng KW, Bagabag A, Corey L, Jerome KR. Use of the MagNA pure LC automated nucleic acid extraction system followed by realtime reverse transcription-PCR for ultrasensitive quantitation of hepatitis C virus RNA. J Clin Microbiol 2004 Sep 42(9) 4130-4136. 

The studies described here demonstrate that bDNA can be used to quantitate mRNA at physiologic levels and that the technology can also be used effectively in cell biology as well as infectious diseases. The bDNA assays do not have the sensitivity of RT-PCR assays that have been described for the same purposes, but bDNA may be better suited for truly quantitative mRNA measurements, since it does not require reverse transcription or amplification of the target sequences. 

Detection of miRs by reverse transcription (RT) and quantitative PCR (qPCR)... 

The selected RNA must be reverse-transcribed into cDNA for further amplification by PCR. Therefore, the RNA is precipitated and subjected to a reverse transcription reaction according to the protocol of the reverse transcriptase supplier. If the RNA concentration is very low (as in the first selection rounds) a coprecipitant like glycogen has to be added to recover the RNA quantitatively. 

Reverse transcription-polymerase chain reaction (RT-PCR) has been widely used for the detection of cytokine gene expression in clinical samples (W18, K5). However, conventional RT-PCR only offers a semiquantitative analysis. Recently, the Perkin-Elmer Corporation (Wellesley, MA) developed the TaqMan cytokine gene expression plate for real-time, in vitro quantitative evaluation of a panel of human cytokine gene expression using fluorescence detection. 

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