Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products.
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 beta-actin). Relative intensities of ethidium-bromide-stained 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 beta-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.
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D004996:Ethidium / Q000737:chemistry*; D015870:Gene Expression; D008040:Genetic Linkage; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D013091:Spermatogenesis / Q000235:genetics*; D013194:Staining and Labeling; D014158:Transcription, Genetic; D014960:X Chromosome
D000199:Actins; D009841:Oligonucleotides; D012313:RNA; D004996:Ethidium