Polymerase real-time

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

Cultures are subject to time delay. New methodology using real-time polymerase chain reaction (PCR) shortens the reporting time. 

Fortin, N. Y. Mulchandani, A. Chen, W. Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli 0157 H7. Anal. Biochem. 2001, 289, 281-288. 

Kuboniwa, M. Amano, A. Kimura, K. R. Sekine, S. Kato, S. Yamamoto, Y. Okahashi, N. Iida, T. Shizukuishi, S. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol. Immunol. 2004,19,168-176. 

Nakamura, T., Sakaeda, T., Ohmoto, N., Tamura, T., Aoyama, N. et al., Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas, Drug Metab. Dispos. 2002, 30, 4—6. 

Abrahamsen HN, Steiniche T, Nexo E, et al. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction. A methodological study on lymph nodes from melanoma patients. J. Mol. Diagn. 2003 5 34-41. 

Badosa E, Trias R, Pares D, Pla M and Montesinos E. 2008. Microbiological quality of fresh fruit and vegetable products in Catalonia (Spain) using normalised plate-counting methods and real time polymerase chain reaction (QPCR). J Sci Food Agric 88(4) 605-611. 

J.R. Uhl, C.A. Bell, L.M. Sloan, M.J. Espy, T.F. Smith, J.E. Rosenblatt and F.R. Cockerill, Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens the Mayo-Roche rapid anthrax test, Mayo Clin. Proc., 77 (2002) 673-680. 

Nelson SM, Ferguson LR, Denny WA (2005) Demonstration by real time polymerase chain reaction that cellular DNA alkylation by novel aminoindoline compounds affects expression of the protooncogene c-myc. Chem Res Toxicol 18(2) 239-248... 

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

Figure 2 Cytokine gene expression in immunopotentiating reconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). PBMC were cultured in the presence or absence of IRIV. On days 1 and 2, culture cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNAs thus obtained were tested in real time polymerase chain reaction assays in the presence of primers specific for the indicated cytokine genes. Source From Refs. 6 and 9.

Fig. 9.4 Establishment of the quantitative methylation-specific polymerase chain reaction (MSP) analytical procedure. The ABI PRISM 7900 HT Sequence Detector system was used to perform real-time polymerase chain reaction (PCR) using MSP primers and bisulfite-modified template DNA. Upper panels Setting up the conditions to obtain the standard curves with 50% (A) or 25% (B) sequential dilution of the template. Lower panels The amplification curves on the left represent P-actin, unmethylated, and methylated MSP products, respectively, for reelin (RELN) (C). Amplification curves were compared at the set threshold before 40 cycles. Amplification curves from various samples are shown in the lower panel right (D)...

Real-time polymerase chain reaction (RT-PCR), microarray... 

Becker, K. Pan, D. Whitley, . B. Real-time quantitative polymerase chain reaction to assess gene transfer. Hum. Gene. Ther. 1999, 70(15), 2559-2566. 

FIGURE3.10 UCP1 mRNA expression levels in epididymal WAT (Okada et al., 2011). Expression of UCP1 mRNA was estimated by quantitative real-time polymerase chain reaction. Relative values were presented as the ratio of UCP1 mRNA to GAPDH mRNA. 

It should be possible to develop either biochemical- or molecular-based tests for resistance in flatworms, as has been successfully used to detect insecticide resistance in insects. However, the necessary information is not available on the mechanism of resistance to be able to design tests of this type for use with trematodes or cestodes. When developed they will hopefully be based on real-time polymerase chain reaction (PCR) or pyrosequencing. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Sims PW, Vasser M, Wong WL, Williams PM, Meng YG. Immunopolymerase chain reaction using real-time polymerase chain reaction for detection. Anal Biochem 2000 281(2) 230-232. 

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