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 involves extracting total RNA from cells or tissues. This RNA, which includes messenger RNA (mRNA), is then reverse transcribed into complementary DNA (cDNA) using either oligo(dT) primers or random primers. Once the cDNA is synthesized, it serves as a template for PCR amplification, allowing researchers to amplify specific genes or quantify gene expression levels.

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Precautions

1. During the experiment, it's crucial to prevent RNA degradation to ensure the integrity of the RNA sample. Special care should be taken during total RNA extraction to avoid mRNA cleavage.

2. To minimize non-specific amplification, a negative control must always be included in the reaction setup. This helps confirm that any observed amplification is due to the target sequence and not contamination or background noise.

3. Internal controls are essential for accurate quantification of target RNA. Commonly used internal references include G3PDH (glyceraldehyde-3-phosphate dehydrogenase) and β-actin. These reference genes help normalize data by accounting for variations in RNA quantity, loading differences, and temperature fluctuations during PCR.

4. It's important to avoid the plateau phase in PCR, 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. Each target sequence may reach its plateau at a different cycle number, so it's recommended to determine this through separate optimization experiments.

5. Contamination with genomic DNA can interfere with results. To prevent this, treat samples with DNase before reverse transcription. Additionally, design PCR primers to span different exons of the gene to reduce potential cross-amplification between genomic DNA and mRNA.