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This approach to study gene expression is universally known as RT-PCR, because of the role of reverse transcriptase (RT) in the synthesis of first-strand cDNA. RT-PCR is that technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by reverse transcriptases, followed by the amplification of the newly synthesized cDNA by standard PCR procedures. Ph.D, in RNA Methodologies (Fourth Edition), 2010 Publisher Summary Two-step reactions are ideal for detection of several messages from a single RNA sample. One-step reactions are easier to set up and ideal for high throughput screening. There are advantages and disadvantages to both methods. Oligo (dT) primers are generally preferred as they hybridize to the 3ʹ poly (A) tails in mRNAs (transcribed gene sequences), whereas random primers prime anything including ribosomal RNA. Oligo (dT), random hexamer or gene-specific primers can be used. During two-step RT-PCR, the synthesized cDNA is transferred into a second tube for PCR. Only sequence-specific primers may be used.
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One-step RT-PCR combines the RT reaction and PCR reaction in the same tube. RT-PCR can be undertaken in one or two steps. The cDNA serves later as a template for exponential amplification using PCR. The RNA template is converted into complementary (c)DNA by the enzyme reverse transcriptase. Reverse transcription (RT)-PCR is used to amplify RNA targets. Morteza Jalali, in Basic Science Methods for Clinical Researchers, 2017 Variations of PCR Method: Reverse Transcription PCR Gene expression profiling is likely to have a major impact on molecular diagnostics in the coming years and will depend on RNA analysis using RT-PCR and possibly high-density arrays. RT-PCR is commonly used in the diagnosis and quantification of RNA virus infections (e.g., human immunodeficiency virus and hepatitis C virus) and the analysis of mRNA transcripts such as those produced by translocations commonly associated with non-Hodgkin's lymphomas, leukemias, and sarcomas.
Transcribe template manual#
RNA extraction kits for both manual and automated RNA purification exist and, when combined with RT-PCR, make RNA analysis in the clinical laboratory virtually as rapid and equally sensitive as PCR-based DNA amplification.
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In RT-PCR, the starting RNA is subsequently degraded, dsDNA is produced, and PCR amplification proceeds in the usual manner. cDNA is not subject to RNase degradation, making it more stable than RNA. The end product is known as complementary DNA (cDNA). Reverse transcriptase is an RNA-dependent DNA polymerase, catalyzing DNA synthesis using RNA as the template. The discovery of retroviral reverse transcriptase in the early 1970s ultimately made RT-PCR possible. RT-PCR uses RNA as starting material for in vitro nucleic acid amplification. Holland, in Cell and Tissue Based Molecular Pathology, 2009 REVERSE TRANSCRIPTION–POLYMERASE CHAIN REACTION