Reverse Transcriptase PCR (RT PCR) Essay

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One of the best characteristics for the functional status of a certain cell is its gene expression pattern. Cells belonging to different tissues, cells in different developmental or metabolic stages, cells under the influence of specific compounds, or cells within a carcinogenic process differ by their gene expression patterns and thus by their mRNA pools. Currently, the most important technique for the accurate quantitation of gene expression is the fluorescent quantitative real-time RT-PCR (Muller et al., 2002a).
Reverse transcription (RT) followed by the polymerase chain reaction (PCR) is the technique of choice to analyze mRNA expression derived from various sources. Real-time RT-PCR is highly sensitive and allows quantification of
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Alternatively, a single polymerase able to function both as an RNA and DNA-dependent DNA polymerase can be used in a ‘one-enzyme/one-tube’ reaction and minimizes hands-on time in addition to the risk of contamination (Bustin, 2000a).
Various variants of this technique are currently applied. The fluorescence signal is either generated by dyes intercalating into dsDNA [SYBR Green] or by hybridization probes [TaqMan or molecular beacons] relying on fluorescence resonance energy transfer (FRET). Typically, the expression of the target gene is analyzed together with a reference gene to normalize the amount of the PCR template and, thus, to enable the calculation of the relative expression level of the target gene (i.e., normalized gene expression). Instead of using a standard curve, the target gene expression levels are calculated relative to the reference. Therefore, it is critical that the reference is a housekeeping gene [such as glyceraldehyde-triphosphate dehydrogenase (GAPDH), β- actin, or an rRNA gene] that is not influenced by the experimental situation. However, it is still a matter of debate whether the expressions of such housekeeping genes are in fact unaffected by a particular experimental setup or not. In parallel, the normalized copy number of any genomic DNA sequence can be determined as well by quantitative real-time RT-PCR (Muller et al., 2002b)

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