Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue.
RNA from brain tissue (in particular human brain) can often only be extracted from fixed material. As brain tissue is very heterogeneous with regard to cell type, it is important to obtain RNA from small samples of identified cells. The aim of this study was (a) to generate expression profiles from small yet homogeneous samples of fixed brain cells in rat and (b) to verify the reliability of these profiles by comparing them with expression profiles obtained from single fresh neurons of the same cell type. Samples (n=12) of 50 rat dentate granule cells were isolated, using Laser Microdissection and Pressure Catapulting, from paraformaldehyde fixed, paraffin embedded tissue or from frozen, ethanol fixed tissue. In addition, RNA was extracted under visual control from individual dentate granule cells (n=12) in hippocampal slices, after electrophysiological recording with patch clamp electrodes. Our data show that RNA was successfully extracted from ethanol fixed sections yielding expression profiles highly comparable to those from non-fixed, single granule cells. RNA extraction from paraformaldehyde fixed, paraffin embedded tissue was less reliable. The present approach validates expression profiling from small amounts of fixed neurons as a powerful tool to investigate molecular processes if fresh tissue is not available.
Qin
Yongjun
Y
Section Neurobiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 320, 1098SM Amsterdam, The Netherlands. qin@science.uva.nl
Heine
Vivi M
VM
Karst
Henk
H
Lucassen
Paul J
PJ
Joëls
Marian
M
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D015870:Gene Expression; D020869:Gene Expression Profiling; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D066298:In Vitro Techniques; D007834:Lasers; D009474:Neurons / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
D012333:RNA, Messenger; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases