Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy

Chiara Vasco, Maurizio Zuccotti, Carlo Alberto Redi, Silvia Garagna

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1173-1177
Number of pages5
JournalMolecular Reproduction and Development
Volume76
Issue number12
DOIs
Publication statusPublished - Dec 2009

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

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