Polymerase chain reaction reverse transcription technique

Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules. 

A reliable method of measuring the ER content in human breast cancer is important for optimal treatment and a qualified estimate of the recurrence-free survival of the patient. The majority of the studies on the expression of ERs, especially ER 3 in human tissues, have been accomplished using RNA techniques such as reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Although the RT-PCR method is an effective tool to describe the presence of a particular gene in the tissue, this approach does not indicate the specific cell that expresses the gene. 

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. 

In addition to natural ribozymes several synthetic ribozymes with new activity have been obtained by in-vitro selection techniques. Starting with a pool of random RNA sequences, molecules with a desired activity can be isolated by successive cycles of activity selection, reverse transcription into DNA, and amplification by polymerase chain reaction [4a,b]. 

Reverse transcription-polymerase chain reaction (RT-PCR), northern and western blotting, and immunoassays have been used for detection of kallerin mRNA and protein in tissue extracts of ovarian, breast, testicular, and prostate tumors. Immunohistochemical techniques have been used for the detection of KLK7 in ovarian tumors and KLKIO in ovarian and testicular tumors. The serum levels of KLK3 (PSA) and KLKll are evaluated by immunoassay. 

Various methods are available for detecting and quantifying gene-expression levels, including northern blots, differential display, polymerase chain reaction after reverse transcription of RNA, and serial analysis of gene expression. These techniques are used primarily to measure the expression levels of specific genes or to screen for significant differences in mRNA abundance. 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

The principle of in vitro selection is governed by a number of the same principles that apply to the Darwinian theory of evolution, as shown in Figure 2. First, the random sequence DNA is prepared by automated solid-phase synthesis. A mixture of four types of nucleotide is added in a stepwise condensation reaction process. When necessary, this DNA library may be converted to an RNA library by in vitro transcription or to a peptide library by in vitro translation. Second, the prepared DNA, RNA, or peptide library is subjected to affinity selection, and the molecules that bind to a target molecule are selected. Because only a very small part of the library is selected in each selection, the selected fraction is then amplified by a polymerase chain reaction (PCR) or a reverse transcription PCR (RT-PCR) technique. Successive selection and amplification cycles bring about an exponential increase in the abundance of the targeting DNA, RNA, or peptide until it dominates the population. 

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