Optimizing Polymerase Chain Reaction (PCR) to Quantify Processed vs. Unprocessed RNA Levels (2025)

Proper mRNA processing, including the addition of a poly-A tail, is essential for transcription stability and export from the nucleus. Disruptions in this process can lead to mis-regulated gene expression and is associated with cancer. We use a Polymerase Chain Reaction (PCR) based method in yeast to assess mRNA processing efficiency while avoiding the use of radioactive materials. mRNA was isolated from yeast and converted to cDNA, then specific primers were used to amplify the cDNA using PCR, and the products analyzed using gel electrophoresis. Comparing band intensity suggests an approximate 1:2 ratio of processed RNA to total mRNA. These results support the use of PCR-based techniques for evaluating mRNA processing efficiency. This method is a safer, more accessible approach for studying RNA processing regulation and provides a foundation for future research into how mRNA processing is affected by various cellular conditions.