| Summary: | Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. • Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity. • Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity. • In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification. Method name: Phenol-chloroform RNA extraction, Keywords: RNA purification, RNA contamination, RNA extraction, RNA purity, Phenol-chloroform extraction, RNA quantification, NRVMs, NRVM, RT-qPCR
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