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Reverse transcription - quantitative polymerase chain reaction (RT-qPCR)  is the gold standard technique for mRNA quantification. It allows the detection of rare transcripts and the observation of small variations in gene expression. 
In this method, RNA is first transcribed into cDNA (complementary DNA) by reverse transcriptase from total RNA or mRNA (messenger RNA). The cDNA is then used as the template for the qPCR reaction. 
RT-qPCR can be performed in a:

- One-step assay - Combines reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. 

- Two-step assay - The reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies. 

These two protocols have several differences between them, all of which are shown in Table.


Quantification of mRNA by RT-qPCR can be:

Absolute - gives the precise copy number of a target mRNA.
 
Relative - expresses the target quantity for an experimental sample as an n- fold difference relative to a calibrator.

Due its several advantages making it a very cost-effective technique, RT-qPCR is used in a variety of applications including:

-Gene expression analysis
-RNA interference (RNAi) validation 
-Microarray validation
-Pathogen detection
-Genetic testing
-Disease research


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Reverse transcription - quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification. It allows the detection of rare transcripts and the observation of small variations in gene expression. In this method, RNA is first transcribed into cDNA (complementary DNA) by reverse transcriptase from total RNA or mRNA (messenger RNA). The cDNA is then used as the template for the qPCR reaction. RT-qPCR can be performed in a: - One-step assay - Combines reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase.
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