Optimized protocol for high-efficiency mitochondrial RNA isolation from fission yeast in log phase and stationary phase.
The fission yeast Schizosaccharomyces pombe (S. pombe) serves as an important model organism for investigating mitochondrial function and gene regulation. However, obtaining sufficient quantity and high-quality mitochondrial RNA (mtRNA) for techniques like Northern blot analysis remains challenging, particularly from stationary-phase cells with rigid cell walls. We developed an improved extraction method by comparing conventional hot-phenol and commercial column-based approaches with modified Enzymatic-Phenol/Chloroform (EPC) protocol, which replaces harsh thermal/liquid nitrogen steps with gentle enzymatic lysis. Additionally, for hard-to-lyse stationary-phase cells, we introduced a specialized OM Buffer to enhance efficiency. The refined EPC protocol achieved substantially higher mtRNA yields without compromising RNA purity. Northern blot analysis confirmed successful isolation of mtRNAs, revealing strong, well-defined signals for mtRNA. This protocol provides a reliable tool for advanced mitochondrial research in S. pombe and other eukaryotes.
Lu Y, Wang G, Shang J