Improved RNA preservation for immunolabeling and laser microdissection.
Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.
Brown
Amanda L
AL
Faculty of Health, School of Biomedical Sciences and Pharmacy, University of Newcastle , Newcastle NSW 2308, Australia.
Smith
Doug W
DW
D000818:Animals; D007150:Immunohistochemistry / Q000379:methods*; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012335:RNA, Ribosomal