Determination of coexisting nuclear transcription rates and cytoplasmic mRNA levels for gonadotropin subunit genes in rat anterior pituitary.
Nuclear run-on transcription assays allow the study of those factors that regulate the expression of a given gene. Because previously published protocols may have been presented in abbreviated form or not developed for nuclei derived from specific tissues, we have established a comprehensive protocol for those who wish to measure transcription of the gonadotropin subunit genes (alpha, LH-beta, FSH-beta) in the anterior pituitary of the rat. The protocol also allows the determination of simultaneously existing cytoplasmic subunit mRNA levels within the same cells. An especially critical step in nuclear run-on transcription assays is the isolation of newly formed labeled mRNA transcripts from the DNA template. In previously published protocols such isolation has been accomplished through rather tedious, multistep methods. To simplify this isolation process, we have evaluated the use of the commercially available product RNAzol. Nascent mRNA transcripts are attached to chromatin and the DNA template; proteinase K and DNase I are typically utilized to disrupt this attachment, thereby facilitating subsequent extraction. We have found that strict adherence to proteinase K and DNase I digestion prior to RNA extraction with RNAzol, followed by two ethanol/NH4OAc precipitations, will enable one to successfully isolate nascent mRNA transcripts free of unincorporated nucleotides. In summary, we present a comprehensive protocol for determining transcription rates and cytoplasmic mRNA levels for the gonadotropin subunit genes in the anterior pituitary of the rat. RNAzol was found to simplify the extraction of newly formed transcripts and should prove applicable with transcription assays utilizing nuclei from a variety of tissue.
O'Conner
J L
JL
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912.
Wade
M F
MF
D000818:Animals; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D003720:Densitometry; D005260:Female; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis / Q000235:genetics*; D037201:Follicle Stimulating Hormone, beta Subunit; D005786:Gene Expression Regulation; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D007986:Luteinizing Hormone / Q000096:biosynthesis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010052:Ovariectomy; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D014158:Transcription, Genetic
D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D012333:RNA, Messenger; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone