Reverse transcription RT-PCR

Reverse transcription (RT)-PCR ISH is based on RT-PCR. The transcripts in a cell are used to make cDNA by reverse transcription, followed by PCR amplification of the cDNA. Details of these methods and representative applications are covered in Chapter 49. 

There are two different approaches to performing the in situ reverse transcription (RT)-PCR technique (22-25). 

Then perform reverse transcription (RT-PCR) assembling four separate reactions as follows 2.5 pi of the recovered RNA, 2 pi P-22 (100 pM), and 6.5 pi DEPC-treated HjO-... 

Figure 2 In ribosome display, mRNA (A) extracted from a cell is converted into a cDNA library (B) is transcribed back into mRNA with no stop codons. Prokaryotic or eukaryotic proteosomes are added and the ribosome then travels down the mRNA (C) translating until it reaches the end of the mRNA molecule (D), where the ribosome halts. With no stop codon, the release factor proteins cannot bind and so the protein, ribosome, and mRNA are physically associated and can be stabilized by high Mg2+ and low temperatures. This complex could then be bound directly to an immobilized natural product (E), the nonbinding library members washed away and the bound members eluted with EDTA (F), which destabilizes the ribosomal complexes by removing Mg2+. The purified sublibrary is converted into cDNA by reverse transcription (RT-PCR) and amplified by regular PCR (B). The/n vitro transcription and translation can be repeated for another round of selection or the cDNA can be analyzed by agarose electrophoresis and/or sequencing.

Figure 4 Gene expression level confirmation for the egr-1, c-fos, and the VDUPl genes. Our cDNA microarray experiments showed that expression levels for egr-1, c-fos, and the VDUPl genes exhibited high degrees of alternations. Northern blotting or reverse transcription (RT)-PCR was, therefore, used to confirm...

As reported in Section 7.3.1, PCR-DGGE has been widely used in food microbiology, and the diversity and dynamics of microbial populations during the manufacturing and ripening of different products have been profiled by means of DNA-based experiments. Reverse transcription (RT)-PCR-DGGE has also been performed, but to a lesser extent, to obtain a picture of the species that are metabolically active at particular sampling instants. 

Bleve, G., Rizzotti, L., DeUaglio, F., Torriani, S. (2003). Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products. Applied and Environmental Microbiology, 69,4116-4122. 

If PCR technology has been developed for the amplification of DNA, it is because the technology relies on thermostable DNA polymerases. For the amplification of RNA by PCR, the RNA should first be converted to cDNA and then be amplified as DNA, a process known as reverse transcription (RT)—PCR. 

Lee, S.H., et al., 2008b. A polymer lab-on-a-ehip for reverse transcription (RT)-PCR based point-of-care ehnical diagnostics. Lab Chip 8 (12), 2121. Available at http //xlink.rsc. org/ DOI=b811131f. 

Detection of miRs by reverse transcription (RT) and quantitative PCR (qPCR)... 

Fig. 5. Selection scheme for the in vitro selection of RNA libraries. The RNA library is subjected to a selection criterion suitable for the enrichment of functionally active sequences. The few selected individual sequences are amplified by reverse transcription (RT) and polymerase chain reaction (PCR). The PCR-DNA is then subjected to in vitro transcription with T7 RNA polymerase. The resulting enriched and amplified RNA library can be used as the input for the next selection cycle. This process is repeated until active sequences dominate the library. At this point, individual sequences can be obtained by cloning and their sequence can be determined by sequencing...

The detection of viraemia can only be carried out by determining the HCV RNA in the PCR test after prior reverse transcription (RT) (K.B. Mullis, 1985). This also works in the incubation phase with patients who are still anti-HCV-negative with normal transaminase values as well as with serum that is still anti-HCV-negative in the acute phase. Thus the following indications are given for the PCR method ... 

Reverse transcription PCR (RT-PCR) is a modification of the standard PCR technique that can be used to amplify mRNA. The first step is to convert isolated mRNA to a complementary DNA (cDNA) molecule using an RNA-dependent DNA polymerase (also known as reverse transcriptase) during a process called reverse transcription (RT). The complementary DNA can be used as any other DNA molecule for PCR amplification. The primers used for cDNA synthesis can be either nonsequence-specific primers (a mixture of random hexa-mers or oligo-dT primers) or sequence-specific primers (Fig. 2.4). Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool. Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules. Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process. 

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. RT-PCR performed on fresh-frozen tissue provides high-quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplification. To attain a more stable RT-PCR amplification from FFPE tissues, it is typical to choose a target that is less than 150 to 200 nt long. 

Obeid and coworkers [128] reported a microsystem fabricated on two glass plates (each 40 x 45 x 0.55 mm), where a continuous channel network was etched into the bottom plate by standard photolithography and wet chemical etching, followed by thermal fusion-bonding of the two plates to form a closed stmcture. This system (see Fig. 14a) used a continuous flow concept to demonstrate functional integration of reverse transcription (RT) and PCR (RT-PCR) with operator selection of the number of amplification cycles to secure the results shown in Fig. 14b. The RT phase of the measurement involved the synthesis of DNA using mRNA templates and was performed before DNA amplification to allow quantification of mRNAs. The integration of RT and PCR processes within a monolithic chip is often problematic as RT components can interfere with the subsequent PCR... 

Often, RNA is available for analysis, rather than DNA. This is the case in cultures of some viruses e.g. the human immunodeficiency vims (HIV), which only contain RNA. PCR, however, is not capable of using RNA as a template and, therefore, amplification does not take place. In order to analyse genetic material of such species, PCR is combined with another enzymatic reaction called reverse transcription (RT). In RT, the enzyme RNA-directed DNA polymerase also known as reverse transcriptase, is used. This enzyme synthesises, or more accurately transcribes, mRNA to its complementary strand of DNA (cDNA). 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

To analyze RNA by PCR an additional step has to be introduced to transcribe RNA into cDNA, which is a more stable single-strand DNA complementary to the targeted RNA. This can be achieved by the reverse transcription (RT) of the template RNA using a reverse transcriptase enzyme. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers for the reverse transcription reaction, whereas in two-step RT-PCR random primers can be used. 

PCR-compatible aqueous chemistiy in the extraction method. The utility of the device for purification of RNA was also verified by extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PC3l. The vrSPE-SPE device reliably yielded a volume reduction for DNA and RNA purifications by the order of 50- and 14-fold, respectively and both were compatible with downstream PC31 analysis. In addition, purification of all samples cmisumed less reagents (by 2.6-fold) than traditional purification methods (Pig. 3). 

Reverse transcriptase (RT) PCR (RT-PCR) can be used to detect RNA in specimens, especially ssRNA viruses. Using the ssRNA as a template, cDNA (complementary DNA) can be synthesized with the enzyme reverse transcriptase, which can further be used as a template in PCR amplification. In two-step RT-PCR, extracted RNAs are first mixed with RT and suitable primer to synthesize cDNA and then followed by adding Taq polymerase and PCR primer pairs for PCR amplification. In one-step RT-PCR, all the ingredients are mixed together and allowed for one cycle of reverse transcription to synthesize cDNA followed by 30 + cycles of PCR amplification. The product can be analyzed by agarose gel electrophoresis. Commercial RT-PCR test kits are available. 

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