Reverse transcription polymerase chain reaction RT-PCR

Mies C. A simple, rapid method for isolating RNA from paraffin-embedded tissues for reverse transcription-polymerase chain reaction (RT-PCR). /. Histochem. Cytochem. 1994 42 811-813. 

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... 

PTT is based on a combination of reverse-transcription polymerase chain reaction (RT-PCR) and linked in vitro transcription and translation (Fig. 3C). This combination of procedures can selectively detect translation-terminating or nonsense mutations. Unfortunately, it does not find missense mutations, which may be etiologic, depending upon location. 

The first binding studies on solubilized membranes from rat brain demonstrated the presence of a low affinity adenosine receptor with characteristics of the A3 subtype (Oliveira et al. 1991). However, in situ hybridization studies in the rat indicated the presence of A3AR mRNA only in the testis (Meyerhof et al. 1991a Zhou et al. 1992 Rivkees 1994) and not in the CNS (Rivkees et al. 2000). Similarly, no expression of A3AR in the brain of mice or in the hippocampi of humans was detected (Rivkees et al. 2000). However, by reverse transcription-polymerase chain reaction (RT-PCR)... 

Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules. 

A reliable method of measuring the ER content in human breast cancer is important for optimal treatment and a qualified estimate of the recurrence-free survival of the patient. The majority of the studies on the expression of ERs, especially ER 3 in human tissues, have been accomplished using RNA techniques such as reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Although the RT-PCR method is an effective tool to describe the presence of a particular gene in the tissue, this approach does not indicate the specific cell that expresses the gene. 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

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... 

Chain reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive... 

Reverse transcription-polymerase chain reaction (RT-PCR), northern and western blotting, and immunoassays have been used for detection of kallerin mRNA and protein in tissue extracts of ovarian, breast, testicular, and prostate tumors. Immunohistochemical techniques have been used for the detection of KLK7 in ovarian tumors and KLKIO in ovarian and testicular tumors. The serum levels of KLK3 (PSA) and KLKll are evaluated by immunoassay. 

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). 

CBl receptor mRNA was detected in the GI tract of the rat, mouse and guinea-pig (Izzo et al. 2003 Storr et al. 2002). In whole gut homogenates from the guinea-pig, CBl receptor and CB2 receptor-like mRNA transcripts were detected, whereas only CBl receptor mRNA was found in the myenteric plexus (Griffin et al. 1997). CBl receptor mRNA was also detected in human colon (Shire et al. 1995). Reverse transcription-polymerase chain reaction (RT-PCR) found both CBi receptor and CB2 receptor mRNA in the rat stomach and mouse small intestine (Izzo et al. 2003 Storr et al. 2002). The expression level of CBi receptor mRNA in the latter was upregulated after treatment with cholera toxin (Izzo et al. 2003). 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

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. 

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