Abstract
BACKGROUND: Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdissection (LCM). Our aim was to optimize the isolation of high-quality RNA from laser capture microdissected fresh frozen prostate tissue on the level of staining and RNA extraction. RESULTS: Cresyl violet and haematoxylin were compared as histological stains for LCM. While RNA quality was similar for cresyl violet (median RIN 7.4) and haematoxylin (median RIN 7.6), tissue morphology was more detailed with cresyl violet as compared to haematoxylin. RNA quality from the following kits was compared: RNeasy(®) Micro (median RIN 7.2), miRNeasy Mini (median RIN 6.6), Picopure(®) (median RIN 6.0), mirVana™ miRNA (median RIN 6.5) and RNAqueous(®)-Micro (median RIN 6.3). RNA quality from microdissected samples with either the RNeasy Micro or miRNeasy Mini kit, was comparable to RNA isolated directly from whole tissue slices (median RIN 7.5, p = 0.09). Isolated RNA from benign and prostate cancer microdissected tissue demonstrated that RNA quality can vary between regions from the same clinical sample. Additionally, RNA quality (r = 0.89), but not quantity (r = 0.69) could be precisely measured with the Agilent Bioanalyzer. CONCLUSIONS: We demonstrate that staining with cresyl violet results in the isolation of high quality RNA from laser capture microdissected tissue with high discriminative morphology. The RNeasy Micro and miRNeasy Mini RNA extraction kits generated the highest quality RNA compared to Picopure, mirVana and RNAqueous with minimal loss of RNA quality during LCM.