Real time RT-PCR

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. 

Ngazoa, E. S., Fliss, I., and Jean, J. (2008). Quantitative study of persistence of human norovirus genome in water using TaqMan real-time RT-PCR. J. Appl. Microbiol. 104, 707-715. 

Lindeman N, Waltregny D, Signoretti S, et al. Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection. Diagn. Mol. Pathol. 2002 11 187-192. 

Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotech Lett 23 275-282... 

Unlike other methods currently employed for quantitative transcript measurements, including cDNA microarrays and real-time RT-PCR, competitive RT-PCR is amenable to quality control, which is critical for clinical diagnostic and pharmaceutical industry applications. Furthermore, microarray approaches are limited to generating snap-shot like profiles, but they do not control for differences in hybridization efficiencies of different gene probes with their corresponding cDNAs. That is, cross comparisons are relative and not absolute. Real-time PCR has gained acceptance recently largely due to the reduced cost associated... 

Pagliarulo V, George B, Beil SJ, Groshen S, Laird PW, et al. 2004. Sensitivity and reproducibility of standardized competitive RT-PCR for transcript quantitation and its comparison with real time RT-PCR. Mol Cancer 3 5... 

Lopez-Errasquin, E., Vazquez, C., Jimenez, M., and Gonzalez-Jaen, M. T. (2007). Real-Time RT-PCR assay to quantify the expression oifuml and fuml9 genes from the fumonisin-producing Fusarium verticillioides. ]. Microbiol. Methods 68,312-317. 

Lindqvist M, Aimer S, Peterson C et al. Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression. Eur J Clin Pharmacol 2003 59 207-211. 

Real-time RT-PCR Determines expression of a particular gene of interest (mRNA) in a cell population Simple, sensitive, quick, specific and quantitative. Lysed cells required Yes... 

Fang, W., and Bidochka, M. J. (2006). Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR. Mycological Research, 110, 1165-1171. 

Strong KL, Rushmore TH, Richards KM. Quantitation of human CYP3A4, 3A5 and 3A7 RNA levels in cultured primary hepatocytes using real-time RT-PCR. ISSX Proc 1999 15 69. 

Medhurst AD, et al. Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR. Brain Res Mol Brain Res 2001 90(2) 125-134. 

F. 1 Constitutive expression of CNS cytokines. RNA from brains of male and female BALB/c mouse pups at 21 days of age was quantified by real-time RT-PCR. Whole-brain RNA was isolated using the Qiagen lipid Tissue Midi RNA isolation kit. Brains were pooled by gender within each litter. Cytokine mRNA quantity was normalized to endogenous control GAPDH. Each bar represents mean S.D. for /V of 3 L. All of the cytokine levels significantly (p < 0.05) differed from each other except the following IL-6 TNF , lL-13 lFNy, and TNF lFNy LT-P from IL-5, IL-6, IL-11, IL-13, TNFa, and IFNy... 

Gene expression of 14 cytokines was measured by real-time RT-PCR in the perfused brain tissue of male and female 21-day-old mouse pups. As shown earlier, there are no significant differences in the expression levels between males and females. Messenger RNA for IL-4, IL-10, and IL-12p40 was not detectable in the... 

Fig. 3 Pb effect on expression of cytokines IL-6, TGF-P1, and IL-18 in the brain. Cytokine mRNA from the brains of female and male mouse pups at pnd21 was quantified by real-time RT-PCR. Whole-brain RNA was isolated using a Qiagen lipid Tissue kit. Mouse pup brains from each litter were pooled according to gender. AU cytokine RNA quantitation results were normalized to endogenous GAPDH. Each bar represents mean S.D. for an iV of 3 L. Significance, indicated by an asterisk,, was determined by the Student s /-test, p < 0.05. The p value for both female and male lL-6 gene expression Pb is 0.056 and the p value for male TGF-P1 gene expression Pb is 0.20 (Data for lL-6 and TGF-beta has been published in Kasten-JoUy et al., J. Biochem. Molec. Toxicol. 2010)...

Liss, B. Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucl. Acids. Res. 2002, 30, e89. 

Verstrepen WA, Bruyriseels P> Mertens AH. Evaluation of a rapid real-time RT-PCR assay for detection of enterovirus RNA in cerebrospinal fluid specimens. 

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). 

M. W. Pfaffl, A new mathematical model for relative qnantification in real-time RT-PCR. Nucleic Acids Res 29(9) e45 (2001). 

Real time RT-PCR High specificity (also according to the serotype) Relatively sophisticated equipment... 

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