This application note describes a method for extracting and isolating total RNA from rodent tissues that have been fixed in formalin and embedded in paraffin, and the subsequent amplification of mRNA from these tissues for use in microarray experiments. We have used this methodology to demonstrate that robust and reliable methods do exist for retrieval of high quality nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues and that these results display a high level of concordance as compared to frozen sample counterparts.
Large numbers of putative drug compounds have been studied in mouse and rat models for their effects on diseases, as well as for their toxicological effects. Enabling high-throughput gene expression analysis of tissues from these models will revolutionize disease diagnoses and drug discovery. Limiting this approach, however, is the fact that these tissues are commonly preserved by Formalin-Fixation and Paraffin-Embedding. Since the macromolecules in FFPE tissues are crosslinked, efficiently extracting and isolating RNA of adequate quality for microarray analysis from such samples poses a significant technical challenge. Arcturus has developed and optimized a novel process that integrates efficient isolation of total cellular RNA from FFPE sections, linear amplification of transcripts from isolated RNA, and synthesis of labeled cRNA for microarray analysis, and incorporated them together in the Paradise Reagent System. Results of a comparison between matched frozen and formalin-fixed samples in order to study the fidelity of gene expression ratios derived from both samples will be presented within this application note.
Equipment, Materials and Reagents
This protocol requires the following reagents for completing the study. For additional reagent and equipment requirements for slide preparation, RNA extraction,