Reverse transcription PCR assays

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. 

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

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. 

Zehentner BK, Dillon DC, Jiang Y, et al. Application of a muitigene reverse transcription-PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes. Clin Chem 2002 48 1225-31. 

Kessler HH, Santner B, Rabenau H, Berger A, Vince A, Lewinski C, et al. Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay. J Clin Microbiol 1997 35 976-7. 

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). 

Doohan EM, Weston G, Rezanoor HN, Parry DW, Nicholson P (1999) Development and Use of a Reverse Transcription-PCR Assay to Study Expression of Tri5 by Fusarium Species In Vitro and In Planta. Appl Environ Microbiol 65 3850... 

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. 

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). 

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. 

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. 

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. 

WalkerPeach CR, Winkler M, DuBois DB, Pasloske BL. Ribonuclease-resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus. Clin Chem 1999 45 2079-85. 

Methods BF = bright field LM of tissue impression smears, wet-mounts, stained whole mounts LM = light microscopy PH = phase microscopy DF = dark-field microscopy TEM/SEM = transmission/scanning electron microscopy of sections or of purified or semi-purified virus ELISA = enzyme-linked immunosorbent assay PAb = polyclonal antibodies MAb = monoclonal antibodies DBH = dot blot hybridization ISH = in situ hybridization PCR = polymerase chain reaction RT-PCR = reverse transcription PCR. 

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. 

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

NIS mRNA such as by northern blotting. Also, through the process of reverse transcription and a polymerase chain reaction (hereafter referred to as RT-PCR), a DNA strand identical to the RNA strand is formed and detected in a very sensitive assay. These RNA-based assays are two methods that can determine whether the symporter mRNA was present in a given tissue. Detection of symporter mRNA would indicate that the gene was being expressed. 

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