Tongke biological RT-PCR experimental principle and precautions - Database & Sql Blog Articles

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Principle of experimental method

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is based on the extraction of total RNA from tissues or cells, using mRNA as a template, and using Oligo (dT) or random primers for reverse transcription. The enzyme is reverse transcribed into cDNA. PCR amplification is performed using cDNA as a template to obtain a gene of interest or to detect gene expression.

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Precautions

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RNA degradation should be prevented during the experiment to maintain RNA integrity. Care should be taken to avoid cleavage of mRNA during total RNA extraction;

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In order to prevent non-specific amplification, a negative control must be provided;

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Setting of internal parameters: mainly for the quantification of target RNA. Commonly used internal references are G3PD (glyceraldehyde-3-phosphate dehydrogenase), β-Actin (β-actin) and the like.

The purpose is to avoid errors caused by RNA quantification errors, loading errors, and temperature differences between the pores in the PCR reaction system.

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PCR cannot enter the plateau phase, and the plateau effect is related to the length, sequence, secondary structure of the amplified target gene and the number of target DNA starts.

Therefore, the number of cycles in which the platform effect occurs for each target sequence should be determined by a separate experiment;

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Prevent DNA contamination: Treat DNA with DNase; where possible, place PCR primers in different exons of the gene to eliminate collinearity between the gene and mRNA.