RT-PCR

RT-PCR
Name	RT-PCR
Caption	Reverse transcription polymerase chain reaction workflow
Classification	Molecular biology technique
Invented	1980s
Inventor	Kary Mullis; Michael Smith; others
Purpose	RNA detection and quantification

RT-PCR Reverse transcription polymerase chain reaction is a laboratory technique that combines reverse transcription of RNA into DNA with polymerase chain reaction amplification. It is widely used in molecular biology, virology, clinical diagnostics, and research to detect and quantify specific RNA sequences. The method underpins diagnostic responses in outbreaks and contributes to research in genetics, oncology, and developmental biology.

Introduction

RT-PCR converts RNA templates into complementary DNA (cDNA) using reverse transcriptase enzymes and then amplifies target sequences through thermal cycling with a DNA polymerase. The technique is central to workflows in institutions such as the Centers for Disease Control and Prevention, World Health Organization, Johns Hopkins University, Harvard University, and private companies like Roche and Thermo Fisher Scientific. Applications span from pathogen surveillance at the United States Centers for Disease Control and Prevention to gene expression studies at universities such as Stanford University and Massachusetts Institute of Technology.

Principles and Methodology

The method begins with RNA extraction from samples collected in clinical settings like Mayo Clinic or research settings at laboratories affiliated with National Institutes of Health and Cold Spring Harbor Laboratory. Reverse transcription uses enzymes derived from organisms studied by researchers at institutions like University of Cambridge and University of California, Berkeley, then polymerase chain reaction, developed by Kary Mullis and refined in companies such as PerkinElmer, amplifies cDNA targets. Oligonucleotide primers and probes designed with software tools influenced by work at European Molecular Biology Laboratory and Broad Institute ensure specificity for genes associated with studies at The Scripps Research Institute or clinical markers used at Mayo Clinic. Thermal cyclers from manufacturers like Bio-Rad Laboratories and detection systems employing fluorescent chemistries devised by teams at DuPont and Applied Biosystems enable quantitative readouts used in collaborations with Bill & Melinda Gates Foundation-funded programs.

Types and Variants

Conventional RT-PCR produces end-point detection similar to assays used in early viral diagnostics developed at Centers for Disease Control and Prevention and Walter Reed Army Institute of Research. Real-time quantitative RT-PCR (qRT-PCR) with intercalating dyes or hydrolysis probes was advanced by groups at National Center for Biotechnology Information and commercialized by Roche and Thermo Fisher Scientific. Digital RT-PCR, leveraging partitioning approaches from work at MIT and Harvard Medical School, allows absolute quantification without standard curves. One-step RT-PCR assays combining reverse transcription and PCR in a single tube are used in high-throughput labs like those at Laboratory Corporation of America and Quest Diagnostics, whereas two-step protocols separating cDNA synthesis and amplification are common in academic settings such as University of Oxford and Yale University for flexible downstream analyses.

Applications

RT-PCR is used for pathogen detection in outbreaks investigated by World Health Organization and Centers for Disease Control and Prevention, including diagnostic assays for viruses studied at Johns Hopkins University Hospital and Mount Sinai Health System. It quantifies gene expression changes in cancer research at Memorial Sloan Kettering Cancer Center and developmental biology studies at European Molecular Biology Laboratory. Environmental monitoring programs run by agencies like United States Environmental Protection Agency and public health labs at Public Health England employ RT-PCR for surveillance of microbial communities. Forensic laboratories at institutions such as Federal Bureau of Investigation use RT-PCR-derived data in molecular analyses, while agricultural research at United States Department of Agriculture and Agricultural Research Service applies the technique to plant pathogen diagnostics.

Limitations and Sources of Error

RT-PCR results can be affected by RNA degradation in samples collected at field sites like those used by Doctors Without Borders or clinical centers such as Cleveland Clinic. Contamination risks arise in multi-laboratory networks exemplified by collaborative projects between Wellcome Trust-funded consortia and national public health laboratories. Primer–dimer formation and nonspecific amplification, issues encountered in assay development at Broad Institute and European Molecular Biology Laboratory, can produce false positives. Assay sensitivity varies with extraction methods compared across studies at Johns Hopkins University and reagent lots from suppliers like Qiagen. Quantification can be biased by reverse transcription efficiency differences first noted in early studies from Cold Spring Harbor Laboratory and by PCR inhibitors reported in environmental sampling projects led by United States Geological Survey.

History and Development

Early concepts combining reverse transcription with polymerase amplification emerged after the invention of reverse transcriptase enzymes studied by teams at Rockefeller University and polymerase chain reaction invented by Kary Mullis at DuPont. The technique's clinical adoption accelerated through standardization efforts involving World Health Organization reference laboratories and regulatory guidance from Food and Drug Administration and European Medicines Agency. Commercialization and scaling were driven by companies including Roche, Thermo Fisher Scientific, Bio-Rad Laboratories, and Applied Biosystems, while methodological refinements originated in academic centers such as University of Cambridge, Massachusetts Institute of Technology, and Stanford University. Ongoing developments in point-of-care RT-PCR devices have involved collaborations among innovators at MIT, startups incubated by Y Combinator, and public health initiatives funded by Bill & Melinda Gates Foundation.

Category:Molecular biology techniques