Reverse transcription quantitative polymerase chain reaction

Godfrey TE, Kim S-H, Chavira M, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5 nuclease quantitative reverse transcription-polymerase chain reaction. J. Mol. Diagn. 2000 2 84-91. 

Macabeo-Ong M, Ginzinger DG, Dekker N, et al. Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses. Mod. Pathol. 2002 15 979-987. 

Bustin SA, Nolan T. 2004. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15 155-166. 

Yajima, T. Yagihashi, A. Kameshima, H. Furuya, D. Kobayashi, D. Hirata, K. Watanabe, N. Establishment of quantitative reverse transcription-polymerase chain reaction assays for human telomerase-associated genes. Clin. Chim. Acta 2000, 290(2), 117-127. 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

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. 

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... 

The 5-HT6 receptors are positively coupled to adenylate cyclase. In the absence of selective agonists and antagonists as well as radioligands, they have been initially localized in the rat brain by Northern blot analyses (218,219), in situ hybridization histochemistry (219-221), and quantitative reverse transcription followed by polymerase chain reaction (222) (see also ref. 210). These studies have established that the receptor mRNA is abundant in extrapyramidal areas such as the striatum, as well as in limbic areas such as the nucleus accumbens, olfactory tubercle, hippocampus, and hypothalamus. 

Badve SS, et al. Estrogen- and progesterone-receptor status in ECOG 2197 comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory. J Clin Oncol 2008 26 2473-81. 

M.M. Wolf, C.R. Ferguson, J. Ibbotson, S.Fl. Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective... 

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). 

G. Smith, R. S. Dawe, C. Clark, A. T. Evans, M. M. Comrie, C. R. Wolf, J. Ferguson, and S. H. Ibbotson, Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation. J Invest Dermatol 121(2) 390-398 (2003). 

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. 

Human cell response to environmental organic toxins. Holman et al. (2000) have investigated the changes in intracellular phosphate and lipid concentrations in human cells as a function of exposure to polychlorinated hydrocarbons and compared their findings to quantitative results from reverse transcription polymerase chain reaction (RT-PCR). The human cells were exposed to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent and well-studied man-made toxin. TCDD causes harmful effects at exposure levels of hundreds to thousands of times lower doses than most chemicals of environmental concern. This polychlorinated hydrocarbon causes an increase in the production of the cytochrome P4501A1 (CYP1A1) gene, which can be monitored with RT-PCR. 

Soong, R., Beyser, K., Basten, O., Kalbe, A., Rueschoff, J., and Tahiti, K., Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes. Clin. Cancer Res. 7, 3423-3429 (2001). 

Ueno, H., Yoshida, K., Hirai, T., Kono, F., Kambe, M., and Toge, T., Quantitative detection of carcinoembryonic antigen messenger RNA in the peritoneal cavity of gastric cancer patients by real-time quantitative reverse transcription polymerase chain reaction. Anticancer Res. 23, 1701-1708 (2003). 

W4. Wong, I. H., Yeo, W., Chan, A. T., and Johnson, P. J., Quantitative relationship of the circulating tumor burden assessed by reverse transcription-polymerase chain reaction for cytokeratin 19 mRNA in peripheral blood of colorectal cancer patients with Dukes stage, serum carcinoembryonic antigen level, and tumor progression. Cancer Lett. 162, 65-73 (2001). 

Molecular methods provide a highly accurate means to assess gene expression for individual components of the PA/PAI system. Northern blot analysis has been widely used in the semiquantitative evaluation of mRNA PA/PAI gene expression (Al, C2, D6, F2, L8, LIO, R7, S7, S8). A 2002 article has described a sensitive reverse transcription-polymerase chain reaction (RT-PCR) method for uPA and PAI-1 mRNA quantitation and demonstrated its usefulness for evaluation of primary breast cancer (Cl). Despite the advantages of molecular technology, quantitative results of gene expression typically require correlation to actual protein mass concentration and/or protein functional activity. 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

Table 2. DMRTl expression in vertebrates. GSD-genetic sex determination, TDS-temperature dependence sex determination, T-testis/genital ridge in male embryo, O-ovary/ genital ridge in female embryo, K-kidney, L-liver, H-heart, M-muscle, LU-lung, S-spleen, ISH-in situ hybridisation, RT-PCR-reverse transcription-polymerase chain reaction, qRT-PCR-quantitative RT-PCR, NB-Northern blot, DB-Dot blot, IHC-immunohistochemistry, WB- Western blot, RPA-RNase protection assay.

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