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Competitive PCR for quantitation of gonadotropin-releasing hormone mRNA level in a single micropunch of the rat preoptic area.
- Source :
-
Molecular and cellular endocrinology [Mol Cell Endocrinol] 1993 Nov; Vol. 97 (1-2), pp. 153-8. - Publication Year :
- 1993
-
Abstract
- A competitive polymerase chain reaction (PCR) for quantitating gonadotropin-releasing hormone (GnRH) mRNA level in a single micropunch of the rat preoptic area (POA) is described. The POA (600 microns in depth) was micropunched from frozen rat brain slices and used for mRNA isolation using Dynabeads-oligo(dT) magnetic separation technique. The target RNA combined with a synthetic, deletion mutant GnRH cRNA as an internal standard, is co-reverse transcribed, and their cDNAs are subsequently co-amplified by Taq DNA polymerase in the same tube in which the same GnRH primers are used. This PCR protocol is sensitive enough to detect GnRH mRNA level in a single POA micropunch derived from an individual rat. There is a linear increase of the amount of GnRH PCR products as a function of input RNA and of the number of PCR cycles. Addition of mutant GnRH cRNA as an internal standard allows us to quantitate GnRH mRNA level in biological samples and to compensate variations of PCR reaction between samples. Following preoptic treatment with 5'-ADMP, which depletes selectively norepinephrine (NE), GnRH mRNA level was significantly reduced. This simple, yet highly sensitive PCR method appears to be a valuable tool for the study of the cellular and molecular regulation of GnRH gene expression in a variety of experimental models.
- Subjects :
- Animals
Base Sequence
Binding, Competitive
Biopsy, Needle
Dopamine analysis
Gene Expression Regulation
Molecular Sequence Data
Norepinephrine analysis
Preoptic Area pathology
Rats
Gonadotropin-Releasing Hormone genetics
Polymerase Chain Reaction
Preoptic Area chemistry
RNA, Messenger analysis
Subjects
Details
- Language :
- English
- ISSN :
- 0303-7207
- Volume :
- 97
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Molecular and cellular endocrinology
- Publication Type :
- Academic Journal
- Accession number :
- 8143898
- Full Text :
- https://doi.org/10.1016/0303-7207(93)90222-6