Together with the reverse transcription (RT) reaction, the PCR method has been widely applied to the quantitative measurement of DNA and RNA messages. BBRC 289, 647–652.Polymerase chain reaction (PCR) is a very powerful tool for qualitative evaluation of nucleic acids due to its high efficiency and convenience. (2001) Real time RT-PCR shows correlation between retinoid-induced apoptosis and NGF-R mRNA levels. Vuillaume, I., Schraen-Maschke, S., Formstecher P., and Sablonniere, B. (2001) High-resolution semiquantitative real-time PCR without the use of a standard curve. Gentle, A., Anastasopoulos, F., and McBrien, N. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔ method. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assay. (1993) Kinetic PCR analysis: real time monitoring of DNA amplification reactions. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. (2001) Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. (1987) Single step method of RNA isolation by acid quanidinium thiocyanate-phenol-chloroform extraction. Cold Spring Harbor Laboratory Press, Plainview, NY.Ĭhomczynski, P. (1989) Molecular Cloning: A Laboratory Manual. (2001) Quantification of murine IFN-(mRNA and protein expression: impact of real-time kinetic RT-PCR using SYBR Green I dye. Hein, J., Schellenberg, U., Bein, G., and Hackstein, H. (1996) Use of elevated reverse transcription reaction temperatures in RT-PCR. (1999) Quantitative RT-PCR: pitfalls and potential. This process is experimental and the keywords may be updated as the learning algorithm improves.įreeman, W. These keywords were added by machine and not by the authors. Indeed, attempts to be absolutely quantitative provide a very significant set of technical concerns ( 1) that are not the subject of the present discussion. The great utility of the technique is that if one has access to a real-time PCR thermocycler and is interested in comparing relative mRNA levels, the experiments are neither difficult nor expensive to perform. Two of the most common uses of this technology in the field of drug abuse are: (1) to compare relative or absolute levels of expression of genes between control and treatment groups or, more recently, (2) for confirmation of gene expression (mRNA) changes identified in a primary screen (e.g., hybridization array analysis). The cDNA, which is much less prone to degradation than RNA, can then be amplified by PCR and quantified to determine the relative abundance of expressed genes within and between sample groups. Starting with a very small amount of material (usually total RNA), the investigator is able to copy the RNA by reverse transcription (RT) to produce single-stranded, complementary DNA (also known as first-strand cDNA). The reverse transcription-polymerase chain reaction (RT-PCR) has become a standard tool in gene expression analysis studies ( 1, 2).

Semi quantitative pcr