Comparison of Tissue Preparation, Handling and Dissociation Methods for Total RNA

Abstract

The extraction and isolation of RNA is an integral part of downstream analyses such as RT‐PCR, RT‐qPCR, Northern blotting, and cDNA library construction. The importance of using pure, intact RNA for these processes is well documented and a critical part of downstream analysis success. It is well known that RNA is sensitive to degradation due to mechanical shear, temperature, storage conditions and freeze‐thawing. Furthermore, RNA is highly susceptible to RNAse degradation following the release of nucleases during the tissue disaggregation process. Thus, proper sample handling during the tissue preparation and homogenization processes is crucial when performing any RNA based assay.Herein, we evaluate the impact of multiple methods for tissue harvesting, storage, storage buffer and mechanical dissociation processes on purified RNA yield and integrity. Multiple, freshly harvested murine tissues, including kidney, liver, heart, and lung were snap frozen in liquid nitrogen and stored without buffer or in an RNA stabilizing reagent at −80°C. Tissues were then dissociated via cryomilling or ambient bead milling and RNA was purified using a silica spin column method. The process above was repeated following one, two and three freeze‐thaw cycles. After each purification, RNA was analyzed on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano kit chip, as per the manufacturers' protocol. Gel images, electrophoretograms and RNA integrity numbers (RIN's) were visualized and analyzed on the 2100 Bioanalyzer Expert software (Agilent).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.