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

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Principle of the Experimental Method

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a widely used molecular biology technique that allows for the amplification and detection of specific RNA sequences. The process begins with the extraction of total RNA from biological samples such as tissues or cells. This RNA contains messenger RNA (mRNA), which serves as the template for reverse transcription. Using either oligo(dT) primers, which bind to the poly(A) tail of mRNA, or random hexamer primers, the enzyme reverse transcriptase converts the mRNA into complementary DNA (cDNA). Once the cDNA is synthesized, it is then amplified using PCR to produce multiple copies of the target gene, enabling its detection or quantification.

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Precautions

1. During the experiment, it is crucial to prevent RNA degradation to maintain its integrity. Special care should be taken during the isolation of total RNA to avoid any damage to the mRNA molecules, as even minor degradation can affect the accuracy of downstream results.

2. To minimize non-specific amplification, it is essential to include a negative control in each experiment. This control helps identify any background noise or contamination that might lead to false-positive results.

3. Internal controls are vital for accurate quantification of target RNA. Commonly used reference genes include G3PD (glyceraldehyde-3-phosphate dehydrogenase) and β-actin. These genes serve as stable internal standards to normalize variations in RNA quantity, loading efficiency, and temperature fluctuations across different wells in the PCR reaction.

4. It is important to avoid reaching the plateau phase during PCR amplification, as this can lead to inaccurate quantification. The plateau effect is influenced by factors such as the length, sequence, and secondary structure of the target gene, as well as the initial number of target DNA molecules. Therefore, the cycle number at which the plateau occurs must be determined for each specific target through preliminary experiments.

5. To prevent DNA contamination, it is recommended to treat RNA samples with DNase before reverse transcription. Additionally, designing PCR primers that span different exons of the target gene can help distinguish between genomic DNA and cDNA, reducing potential cross-amplification issues.