Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: Ethidium-bromide staining of RT-PCR products

John R. McCarrey, Donald D. Dilworth, R. Mark Sharp

Research output: Contribution to journalArticlepeer-review

53 Scopus citations

Abstract

We have used analysis of ethidium-bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products to assess the effects of X-chromosome inactivation during spermatogenesis in the mouse. RT-PCR was performed on total RNA from eight different spermatogenic cell types, including premeiotic spermatogonia, meiotic spermatocytes, and postmeiotic spermatids, to detect transcripts from five different X-linked structural genes (Pgk-1, Zfx, Pdha-1, Hprt, and Phka) and two autosomal genes (Pgk-2 and β-actin). Relative intensities of ethidium-bromide-strained RT-PCR products representing transcripts from each gene in each cell type were analyzed by densitometry using the Image program (version 1.4, NIH), and normalized against β-actin values. These results suggest a coordinate inactivation of the X-linked loci at the onset of meiosis, followed by variable rates of decline of corresponding transcript levels reflecting differential mRNA stabilities and/or leaky expression after inactivation. Technically, these results indicate that analysis of ethidium-bromide-stained RT-PCR products can be used to provide a "semiquantitative" indication of relative levels of specific transcripts in a developing cell lineage without using radioactive probes to quantitate these products.

Original languageEnglish (US)
Pages (from-to)117-123
Number of pages7
JournalGene Analysis Techniques
Volume9
Issue number4
DOIs
StatePublished - Aug 1992
Externally publishedYes

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Genetics

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