Reverse transcription-PCR

Baert, L., Wobus, C. E., Van Coillie, E., Thackray, L. B., Debevere, J., and Uyttendaele, M. (2008c). Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Appl. Environ. Microbiol. 74, 543-546. 

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. 

La Rosa, G., Fontana, S., Di Grazia, A., laconelli, M., Pourshaban, M., and Muscillo, M. (2007). Molecular identification and genetic analysis of Norovirus genogroups I and II in water environments Comparative analysis of different reverse transcription-PCR assays. Appl. Environ. Microbiol. 73,4152M161. 

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. 

Sheridan, G. E. C. Masters, C. I. Shallcross, J. A. Mackey, B. M. Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells. Appl. Environ. Microbiol. 1998, 64,1313-1318. 

Quadroni M et al. Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAOl. Eur J Biochem 1999 266 986-996. 

Ripe tomato fruits accumulate significant amounts of lycopene, but only trace amounts of xanthophylls. Dharmapuri and others (2002) overexpressed the lycopene (3-cyclase (b-Lcy) and (3-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter, and transgene and protein expression was followed through semiquantitative reverse- transcription PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of (3-carotene, (3-cryptoxanthin, and zeaxanthin the carotenoid composition of leaves remained unaltered, and the transgenes and the phenotype were inherited in a dominant Mendelian fashion. 

Gettemy JM, Ma B, Alic M, Gold MH (1998) Reverse transcription PCR analysis of the regulation on the manganese peroxidases gene family. Appl Environ Microbiol 64(2) 569-574... 

Nishimura M, Yaguti H, Yoshitsugu H, Naito S, Satoh T (2003) Tissue distribution of mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 123(5) 369-375. 

McGrath, S., Dolley, J.S.G. and Haylock, R.W., Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription PCR, Appl. Env. Microbiol., 66, 1423-1428, 2000. 

The vast majority of the smdies on antibiotic resistance in the environment have focussed on the survey of resistance genes. However, the mere detection of the antibiotic resistance genes may be insufficient to get a clear perspective of their function in the environment. If the role of antibiotic resistance genes in the environment is to be assessed, it is also important to determine the factors capable of triggering gene expression and to measure the expression levels [24]. Such an approach requires transcriptomic analyses supported, for instance, by reverse-transcription PCR or microarrays. 

AGE — agarose gel electrophoresis, BAL = bronchoalveolar fluid, FAM = 6-carboxyfluorescein label, MGB = minor groove binder, n.s. - not specified, RAPD = randomly amplified polymorphic DNA, RT-PCR = reverse transcription PCR, TAMRA = 6-carboxytetramethylrhodamine label. 

Doohan, F. M., Weston, G., Rezanoor, H. N., Parry, D. W., and Nicholson, P. (1999). Development and use of a reverse transcription PCR assay to study expresdion of tri5 by Fusarium species in vitro and in planta. Appl. Environ. Microbiol. 65, 3850-3854. 

Ali, N. Jameel, S. (1993). Direct detection of Hepatitis C virus RNAin serum by reverse transcription PCR. Bio/Techniques, 15, 40-2. 

Gardner, R.J., Bobrow, M., and Roberts, R.G., 1995, The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test, Am J Hum Genet, 57, pp 311—320. 

A role for DOR genes in olfaction requires that they are expressed in OSNs, where they can interact with odors and transmit odor-activated neuronal activity. To address this question Clyne et al. (1999) used reverse transcription-PCR (RT-... 

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... 

List of Abbreviations CD/MRV, Common Disease/Multiple Rare Variants DAAO, D-amino acid oxidase DAOA, D-aminoacid oxidase activator EST, expression sequence tag GEO, Gene Expression Omnibus MAF, minor allele frequency NIMH, National Institute of Mental Health NMDA, N-methyl D-aspartate RT-PCR, reverse transcription-PCR... 

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. 

Foster, R. A., CoUier, J. A., et al. (2006). Reverse transcription PCR amplification of cyanobacteria symbiont 16S rRNA sequences from single non-photosynthetic eukaryotic marine planktonic host cells. J. Phycol. 42, 243-250. 

Nogales, B., Timmis, K. N., NedweU, D. B., and Osborn, A. M. (2002). Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA. Appl. Environ. Microbiol. 68, 5017—5025. 

Griffin JL, Blenkiron C, Valonen PK, Caldas C, Kauppinen RA. High-resolution magic angle spinning IH NMR spectroscopy and reverse transcription-PCR analysis of apoptosis in a rat glioma. Anal. Chem. 2006 78 1546-1552. 

Like all laboratory procedures, the PERT assay requires adequate controls to assure the quality of the entire detection procedure. Negative and positive controls must be included for each step of the procedure (i.e., sample preparation, reverse transcription, PCR amplification, and detection of amplified product). In addition, to quantify RT, the PERT assay requires an activity standard. 

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