期刊
MOLECULAR AND CELLULAR ENDOCRINOLOGY
卷 429, 期 C, 页码 93-105出版社
ELSEVIER IRELAND LTD
DOI: 10.1016/j.mce.2016.04.001
关键词
StAR; Transcription; Splicing; Fluorescence in situ hybridization; PCR
资金
- NIH [RO1 DK074819, RO1 DK090249]
The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
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