RNA Analysis Prior to Use in Microarrays

RNA Analysis Prior to Use in Microarrays

Miniaci Performing Arts Center

Extracting RNA from an organism and isolation of mRNA is an intermediate step done prior to its use in further applications. Therefore, assessment of RNA presence, including its quality is important. There are many techniques that are currently available; however, they can be time consuming or sample consuming. The purpose of this experiment was to determine an efficient way to assess RNA prior to using the sample in a microarray-based study. Twenty hours after Saccharomyces cerevisiae was plated on YEPD media, RNA was extracted using Ambion’s RiboPure Yeast Kit. Different protocols were examined to determine an efficient way to analyze the RNA. First, absorbance readings at 260nm and 280nm were obtained. NEBioLabs’ Protoscript First Strand cDNA Synthesis Kit was used to synthesize cDNA from mRNA via Reverse Transcriptase Polymerase Chain Reaction. PCR was also used to amplify TDH1, a housekeeping gene present in mRNA. These products were subsequently analyzed for RNA presence using different electrophoresis gel protocols, including the use of denaturing buffers and prepackaged gels. The presence of bands at 18s and 28s confirmed original RNA presence. In addition to spectrophotometry for qualitative analysis, it was determined that electrophoresis of the extracted RNA, treated with Superload, a denaturing buffer, was efficient in verifying the actual presence of RNA.