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RT-PCR. Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). [1] It is primarily used to measure the amount of a specific RNA.
The sample is mixed with the primers, reverse transcriptase and DNA polymerase and the reaction takes place under a constant temperature. The required temperature can be achieved using a simple hot water bath. PCR requires thermocycling; RT-LAMP does not, making it more time efficient and very cost effective. [3]
The protocols for 5' or 3' RACES differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription) reaction using an anti-sense (reverse) oligonucleotide primer that recognizes a known sequence in the middle of the gene of interest; the primer is called a gene specific primer (GSP). The ...
By adding a reverse transcriptase enzyme to an RPA reaction, it can detect RNA as well as DNA, without the need for a separate step to produce cDNA. [2] [3] [4] Because it is isothermal, RPA can use much simpler equipment than PCR, which requires a thermal cycler. Operating best at temperatures of 37–42 °C and still working, albeit more ...
Two-tailed PCR uses a single primer that binds to a microRNA target with both 3' and 5' ends, known as hemiprobes. [11] Both ends must be complementary for binding to occur. The 3'-end is then extended by reverse transcriptase forming a long cDNA. The cDNA is then amplified using two target specific PCR primers.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) with reverse transcriptase.
Template-switching polymerase chain reaction (TS-PCR) is a method of reverse transcription and polymerase chain reaction (PCR) amplification that relies on a natural PCR primer sequence at the polyadenylation site, also known as the poly(A) tail, and adds a second primer through the activity of murine leukemia virus reverse transcriptase. [1]
Although RNA can also be amplified by PCR using a reverse transcriptase (in order to synthesize a complementary DNA strand as a template), NASBA's main advantage is that it works under isothermal conditions – usually at a constant temperature of 41 °C or two different temperatures, depending on the primers and enzymes used. Even when two ...
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