Efficient Cell Lysis Method for Isolation of Total RNA from Slime Mold Dictyostelium: Applicability in Preparation of cDNA

Dictyostelium is a model organism for understanding of many cellular processes, evolutionary studies, behavioral analysis, gene expression and now even drug metabolism. RNA analysis is vital to most studies at molecular level. Extracting RNA is no easy task due to the omnipresence of robust RNases. Attaining full length mRNAs is the chief task in isolation of pure RNA. Stable ribonucleases are the major obstacle in isolating RNA of any kind. Many methods and kits are available for different organisms with differing efficiency, utility and expense. For isolation of RNA from Dictyostelium, Blumberg and Lodish devised a simple, in-expensive yet effective method, though for identification of mRNA use of DNase and poly-dT columns were required. We modified the cell homogenization process and isolated total RNA with the method. The modification enables us to detect specific mRNA from the total RNA pool without purifying through poly-dT columns or processing for DNA removal. The integrity of RNA isolated by the modified method was similar to that of RNA extracted with Qiagen RNAasy Mini kit indicative of the quality of the total RNA extracted. With this modification, we have been able to identify three mRNA transcripts and specifically amplify the cDNA using sequence specific primers.