The principle is: extract total RNA from tissues or cells, use the mRNA in it as a template, and use Oligo (dT) or random primers to reverse transcriptase into cDNA. Then use cDNA as a template for PCR amplification to obtain the target gene or detect gene expression.
This technology is mainly used to: analyze gene transcription products, obtain target genes, synthesize cDNA probes, and construct efficient RNA transcription systems.
RT-PCR (Reverse Transcription-Polymerase Chain Reaction) is a technology that combines RNA reverse transcription (RT) and cDNA polymerase chain amplification (PCR).
First, cDNA is synthesized from RNA through the action of reverse transcriptase, and then the cDNA is used as a template to amplify and synthesize the target fragment. RT-PCR technology is sensitive and versatile and can be used to detect gene expression levels in cells, the content of RNA viruses in cells, and to directly clone cDNA sequences of specific genes.
The RNA used as template can be total RNA, mRNA or in vitro transcribed RNA product. Regardless of the type of RNA used, the key is to ensure that the RNA is free of RNase and genomic DNA contamination.
The primers used for reverse transcription can be selected from random primers, Oligo dT and gene-specific primers according to the specific conditions of the experiment. For short eukaryotic cell mRNAs that do not have a hairpin structure, all three are available.
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