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Identification, isolation, and RT‐PCR analysis of single stage‐specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy
Author(s) -
Vasco Chiara,
Zuccotti Maurizio,
Redi Carlo Alberto,
Garagna Silvia
Publication year - 2009
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.21086
Subject(s) - biology , spermatogenesis , microdissection , seminiferous tubule , laser capture microdissection , population , microbiology and biotechnology , single cell analysis , housekeeping gene , transillumination , gene expression , andrology , gene , cell , genetics , pathology , sertoli cell , medicine , demography , sociology , endocrinology
The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated spermatids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro‐transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh , an endogenous housekeeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells. Mol. Reprod. Dev. 76: 1173–1177, 2009. © 2009 Wiley‐Liss, Inc.