Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.13/807391

OpenURL Link
Title
Improved RNA preservation for immunolabeling and laser microdissection
Author/Creator
Brown, Amanda L.;  Smith, Doug W.
Institution
The University of Newcastle. Faculty of Health, School of Biomedical Sciences and Pharmacy
Description
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.
Relation
RNA: A Publication of the RNA Society Vol. 15, Issue 12, p. 2364-2374
Publisher Link
http://dx.doi.org/10.1261/rna.1733509
Date
2009
Publisher
Cold Spring Harbor Laboratory Press
Keyword(s)
RNA degradation;  transcriptome preservation;  quantitative PCR;  microfluidics;  capillary electrophoresis
Resource Type
journal article
Identifier
http://hdl.handle.net/1959.13/807391
Identifier
ISSN:1355-8382
Reviewed
Reviewed
28 Visitors 34 Hits 0 Downloads
Save/E-mail Citation
Citation Format
E-mail Address
Subject
"RNA: A Publication of the RNA Society"
 
OR