Application of laser-capture microdissection to analysis of gene expression in the testis.
The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.
Sluka
Pavel
P
Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia.
O'Donnell
Liza
L
McLachlan
Robert I
RI
Stanton
Peter G
PG
D000818:Animals; D002469:Cell Separation; D015870:Gene Expression; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000295:instrumentation* / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013737:Testis / Q000166:cytology* / Q000378:metabolism
D012333:RNA, Messenger