Reverse transcriptase polymerase chain reaction (RT-PCR) has become one of the most widely applied techniques in biomedical research. The ease with which the technique permits a specific mRNA detection and quantification has been a major asset in the molecular investigation of disease pathogenesis. Disease imbalances in the expression of specific mRNAs can be sensitively and quantitatively determined by RT-PCR. Reverse transcriptase polymerase chain reaction also offers some opportunities in diagnostic methods such as the detection of RNA viruses. Different studies have shown the possibility of analyzing RNA extracted from autopsy formalin-fixed, paraffin-embedded tissues[5'10] by RT-PCR analysis. In most of the protocols RNA is firstly transcribed in cDNA and subsequently amplified by PCR. The major concern related to archive postmortem tissues is the enhanced degradation of RNA. Usually, in routinely treated formalin-fixed material of biopsy or surgery origin the amenable fragments of RNA range between 100 and 200 bp. Analysis of RNA obtained from postmortem formalin-fixed, paraffin-embedded tissues showed a higher level of degradation (Table 2). As reported in Fig. 2, amplicons that had been successfully amplified were not longer than 75-100 bp (Fig. 2A and B). In fact, for fragments nearly 150 bp long, the RNA amplification was obtained only in a few samples (Fig. 2C and D). To obtain a successful RT-PCR analysis and maximize the amplification of degraded RNA it is better to amplify short sequences, of about 70-100 bp. It is also possible to perform quantitative RT-PCR analysis using RNAs obtained from necropsy formalin-fixed and paraffin-embedded tissues. Several variables need to be controlled in gene-expression analysis, such as the amount of starting material, enzymatic efficiencies, and differences between tissues. The quantification of RNA from postmortem formalin-fixed, paraffin-embedded tissues presents specific problems that cannot be resolved with a competitive analysis because of RNA degradation in the preserved samples. Because the degradation is presumably random, the quantification in these tissues cannot be resolved using competitive analysis with internal standards. The comparison of different template preparations (standard vs. pathological template) introduces errors. The sample fragmentation due to fixation severely distorts the absolute quantification. In relative quantification the level of the mRNA of interest is related to the amounts of housekeeping genes; these are supposed to be constant both in the tissue and in the pathological situation of interest. As a result, relative quantification is preferred and strongly recommended in postmortem archival tissues. Performing a relative calculation eliminates all distortions due to fixation artefacts or sample impurity. Accurate normalization of gene-expression levels is an absolute prerequisite for reliable results. The purpose of normalization is to remove the sampling differences in order to identify real gene-specific variation. For RT-PCR there is a
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