Improved method for isolating high-quality RNA from mouse bone with RNAlater at room temperature

Abstract

Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR.