Polymerase chain reaction quantitative analysis

Fasco MJ, Treanor CP, Spivack S, Figge HL, Kaminsky LS (1995) Quantitative RNA-polymerase chain reaction—DNA analysis by capillary electrophoresis and laser induced fluorescence. Anal Biochem 224 140-147. 

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. 

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. 

Gilliand G, Perrin S, Blanchard K, Bunn HF. 1990b. Analysis of cytokine mRNA and DNA detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad... 

Polymerase chain reaction (PCR), on the other hand, has several advantages it can be used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffin-embedded tumor tissue can be used, and it can be automated and standardized. Quantitative PCR techniques are currently being assessed for their clinical application to HF.R-2 DNA testing (Vona et al., 1999). However, presently the PCR technology is not optimally suited for routine, clinical application (see next section for details of quantitative analysis of HER-2Ineu expression). 

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. 

The authors would like to thank Dr. Thomas Rushmore, Dr. Karen Richards, and Ms. Kristie Strong-Basalyga for their contribution in the development of the quantitative real-time reverse transcriptase-polymerase chain reaction assay for CYP analysis in primary hepatocytes. 

Biodistribution analysis is conducted at the molecular level. The current gold standard is a quantitative polymerase chain reaction (Q-PCR) assay that detects the number of vector copies per microgram of genomic DNA. Biological fluids and tissue samples are carefully harvested (to avoid crosscontamination) from control and gene therapy product-injected animals at... 

Direct immunohistochemical analysis of prostatic tissue has become very popular since the development of AR antibodies. However, a disadvantage of this technique in quantitative analysis is that the intensity of the immunohistochemical stain is dependent on the intactness of the structure of the AR. Therefore, mutations or alterations in the structure may reduce staining intensity (T5). Biochemical and immunohistochemical studies of AR content in relation to grade or stage of disease, as well as prediction of response to endocrine therapy, has been inconsistent. Nearly all primary prostate cancer specimens positively express AR protein, as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis as well as by immunohistochemical analysis on formalin-fixed, paraffin-embedded primary prostate tissues (D12, H14). In advanced-stage prostate cancer, immunohistochemical techniques has shown that metastases in bone, the... 

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

Landgraf, A., Reckmann, B., and Pingoud, A. (1991) Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a flourescent dye. Anal. Biochem. 193, 231-235. 

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). 

Polymerase chain reaction (PCR) techniques allow one to amplify small quantities of target DNA typically by 10" to 10 times and determine in a semi-quantitative manner the presence of specific microorganisms. PCR analysis can be automated and provide turnaround times on the order of hours rather than the 3 to 5 days required for culture techniques (Atlas, 1991). Quantitation of PCR is accomplished by most probable number methods or by calibration with the function = C ,j i x 2", where C is the concen-... 

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