Improved RNA preservation for immunolabeling and laser microdissection

Abstract

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.

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

“…Finally, in these conditions, the addition of RNAse inhibitor in all solutions is effective in preserving RNA from being degraded. The use of high salt conditions applied during antibody incubation has been previously shown to be effective in preserving RNA 9,10 . However, this alternative method remains limited to quick staining using robust antibodies.…”

“…This approach has been previously performed and briefly described in few publications 1,[9][10][11][12][13] . Here, we demonstrate a detailed procedure to obtain high-quality RNA from a specific subset of cells in a complex tissue structure by combining quick immunolabeling with LCM.…”

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

“…The short incubation times are due to the use of high concentrations of primary and secondary antibodies, concentrations that are around 10-20 fold higher than what is necessary for conventional immunohistochemistry with a 2 h incubation. If however, longer incubation times are required, Brown and Smith used high salt buffers to inhibit RNA degradation and obtain good quality RNA with long incubation times (up to 20 h) 13 . This approach can also be used if there is substantial time required between labeling of the tissue and getting access to a LCM system.…”

Age-related gene expression changes in substantia nigra dopamine neurons of the rat

“…Briefly, four equidistant slides along the rostral-caudal extent of the SN were selected, the tissue sections rinsed in 1xPBS and fixed in acetone. Following fixation, sections were incubated in 2 M NaCl PBS to improve mRNA stability (Brown et al, 2013;Brown and Smith, 2009). Sections were incubated overnight at 4 • C in mouse anti-Th monoclonal antibody (Millipore, Australia) diluted 1:100 in 2 M NaCl PBS.…”

“…1) performed in high salt buffer to maximise RNA preservation (Brown and Smith, 2009). Cells positive for both Th immunofluorescence and the nuclear stain Hoechst, were laser microdissected and catapulted into RNA lysis buffer containing carrier RNA.…”