Modified Method for Competitive Reverse Transcription Polymerase Chain Reaction for Rapid and Automated Quantitation...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Conventional competitive reverse transcription polymerase chain reaction (RT-PCR) is labor-intensive, requiring multiple reactions per sample for messenger RNA (mRNA) quantitation.
• Existing methods involve serial dilutions of internal competitor sequences, making high-throughput analysis challenging.

Purpose of the Study:

• To develop a modified, automated competitive RT-PCR method for efficient and reproducible mRNA quantitation.
• To reduce the number of reactions required for analyzing multiple samples and RNA transcripts.

Main Methods:

• An RNA mimic (competitor RNA molecule) was synthesized and used in conjunction with a fluorescently labeled primer for PCR amplification of target mRNA and the mimic.
• PCR products were analyzed using automated laser-scanned gel electrophoresis, with quantitation based on the area under the curve (AUC).
• A standard curve was generated using known dilutions of standard RNA, and unknown samples were quantified by interpolation.

Main Results:

• The modified method successfully quantified transforming growth factor beta-1 (TGF-beta-1) mRNA in human skin fibroblasts.
• The assay demonstrated high reproducibility with inter- and intra-assay coefficients of variation below 10%.
• The automated process significantly decreased the number of PCR reactions needed per sample.

Conclusions:

• The modified competitive RT-PCR is a highly reproducible technique for accurate mRNA quantitation.
• This automated approach simplifies the analysis of multiple samples and RNA transcripts.
• The method can be adapted to assay multiple RNA molecules simultaneously by generating differentially sized PCR products.