TY - JOUR
T1 - Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse
T2 - Ethidium-bromide staining of RT-PCR products
AU - McCarrey, John R.
AU - Dilworth, Donald D.
AU - Sharp, R. Mark
PY - 1992/8
Y1 - 1992/8
N2 - 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.
AB - 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.
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U2 - 10.1016/1050-3862(92)90051-6
DO - 10.1016/1050-3862(92)90051-6
M3 - Article
C2 - 1282026
AN - SCOPUS:44049115761
SN - 1050-3862
VL - 9
SP - 117
EP - 123
JO - Gene Analysis Techniques
JF - Gene Analysis Techniques
IS - 4
ER -