In-depth comparison of methods to isolate RNA from common human cell lines.

RNA isolation is a fundamental procedure that is universally applied in biomedical research. The isolation of high-quality and high-purity RNA is essential for many biochemical analyses, including RT-qPCR and RNA sequencing. However, this isolation can be challenging due to the instability of RNA, its structural similarity to DNA, and the complexity of biological samples. Numerous methods and kits have been developed and marketed to surmount these challenges. To this day, most studies comparing these methods for RNA isolation have focused on samples that are especially difficult to process, such as FFPE tissue. In contrast, few studies have compared different RNA isolation procedures using simpler samples, such as mammalian cell cultures. Moreover, most studies employ outdated and/or incomplete analysis techniques or only compare a minimal set of methods. This article presents a thorough comparison of various manual RNA isolation methods, with a focus on spin column-based kits. The isolated RNAs were evaluated regarding yield, integrity, and purity using state-of-the-art techniques: photometric analysis, automated electrophoresis-based integrity measurement, and qPCR. While most kits performed satisfactorily, our results highlight some outliers, particularly regarding genomic DNA contamination and RNA integrity. This comprehensive analysis provides researchers with valuable data to support selecting an appropriate RNA isolation method.