Quantitative PCR real-time

The PCR technique is very useful during all stages of the research and development of biotech crops. PCR analysis is used for gene discovery, event selection, screening, transformant identification, line selection and plant breeding. Quantitative real-time PCR is used to determine the number of transgene copies inserted in experimental... 

Fig. 11.3 Effect of HU on ET-1 mRNA expression in the TrHBMEC (a) and EA-hy 926 (b) endothelial cells in culture. Quantitative real-time PCR was used to assess the level of ET-1 mRNA in at least four independent experiments in duplicate. Results are expressed in percentage of residual ET-1 mRNA expression for HU-treated cells as compared to the control (culture with or without cytokines). The TATA-binding protein mRNA was used as an internal control. The abbreviations are the same as in the legend for Figure 11.2.

Mule, G., Susca, A., Logrieco, A., Stea, G., and Visconti, A. (2006). Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes. Int. J. Food Microbiol. Ill, S28-S34. 

Pusterla, N. Huder, J. B. Leutenegger, . M. Braun, U. Madigan, J. E. Lutz, H. Quantitative real-time PCR for detection of members of the Ehrlichia phagocyto-phila genogroup in host animals and Ixodes ricinus ticks. J. Clin. Microbiol. 1999, 37(5), 1329-1331. 

Huser, D., Weger, S. and Heilbronn, R. (2002). Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR. J. Virol. 76, 7554—7559. 

Tuzmen, S., Kiefer, J., and Mousses, S. (2007) Validation of short interfering RNA knockdowns by quantitative real-time PCR. Methods Mol Biol. 353 177-203. 

Gentle A, Anastasopoulos F, McBrien NA. High-resolution semi-quantitative real-time PCR without the use of a standard curve. Biotechniques 2001 31 502, 504-506, 508. 

Switching on/off genes mRNA patterns via DNA microarrays, PCR, quantitative real time PCR... 

Kamphuis, W., Schneemann, A. van Beek, L. M., et al. (2001) Prostanoid receptor gene expression profile in human trabecular meshwork A quantitative real-time PCR approach. Investigative Ophthalmology Visual Science, 42, 3209-3215. 

The accuracy of real-time PCR quantification depends not only on the method chosen to analyze the curves, but also on the quality of calibrators used. Purified PCR products quantified by spectrophotometry are easily obtained. When serially diluted, these calibrators can accurately quantify the amount of target in human genomic DNA. Alternatively, purified plasmids or genomic DNA can be used as calibrators. Limiting dilution analysis to determine the amount of ampMable DNA is seldom necessary. The precision of quantitative real-time PCR depends on the copy number. When the initial target concentration is low, imprecision is high. Part of the variance comes from stochastic limitations as defined by the Poisson distribution as described earlier. In addition, the PCR efficiency may be more variable at low copy numbers. 

Conventional cytogenetics, FISH, and RT-PCR have been reliably used for the laboratory detection of the t(9 22). RT-PCR detection is possible in up to 95% of cases, and may detect up to 10% of cases missed by conventional cytogenetics, and is an important modality for minimal residual disease detection. Recently, quantitative real-time PCR-based approaches have improved the ability to detect and quantify BCR-ABL transcripts in CML patients (Figure 39-15 Color Plate 4]). The recent availability the tyrosine kinase inhibitor imatinib mesylate for CML is an important development in the treatment of CML and further underscores the importance of methods for sensitive and specific identification and quantification of the BCR-ABL fusions in patients with a clinical suspicion of a CML. 

Results from gene array experiments should be verified by other mRNA quantification methods such as Northern blots3,4, ribonuclease protection assays8,36,128 dot and slot blots72115, or quantitative real time PCR (Q RT-PCR)58. 

Quantitative Real-Time PCR for Titering (Method Outlined in Subheading 18.3.5)... 

---