Optimizing a Method to Quantify Cellular RNA Levels (2023)

Within a Eukaryotic cell, mRNA is transcribed from DNA in the nucleus, and thereafter translated into proteins within the ribosomes attached to the endoplasmic reticulum (ER). Before translation, mRNA experiences a series of changes. RNA polymerase transcribes the DNA, producing pre-mRNA. The pre-mRNA is then cleaved and polyadenylated (severing the end of the pre-mRNA and adding a series of adenine nucleotides to form a tail-like structure), thereby producing mature mRNA ready for translation into proteins. Improperly processed or unprocessed RNA can produce dysfunctions that may foster development of diseases, such as cancers. We aim to optimize a method to measure quantities of processed and unprocessed RNA. RNA is isolated from yeast cells and then reverse-transcribed to produce cDNA of varying sizes. cDNA levels are analyzed through a Polymerase Chain Reaction (PCR) to quantify ratios between processed and unprocessed RNA. Optimizing this technique is essential for future advancements in potential RNA therapies.