Competitive PCR for quantitation of gonadotropin-releasing hormone mRNA level in a single micropunch of the rat preoptic area

K. Kim, H. Jarry, I. Knoke, Jae Young Seong, S. Leonhardt, W. Wuttke

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 μm 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.

Original languageEnglish
Pages (from-to)153-158
Number of pages6
JournalMolecular and Cellular Endocrinology
Volume97
Issue number1-2
Publication statusPublished - 1993 Nov 1
Externally publishedYes

Fingerprint

Preoptic Area
Polymerase chain reaction
Gonadotropin-Releasing Hormone
Rats
Polymerase Chain Reaction
Messenger RNA
Complementary RNA
Taq Polymerase
RNA
Magnetic separation
DNA-Directed RNA Polymerases
Reaction products
Gene expression
Brain
Norepinephrine
Theoretical Models
Complementary DNA
Gene Expression

Keywords

  • (GnRH)
  • (PCR)
  • Competitive polymerase chain reaction
  • Gonadotropin-releasing hormone
  • Micropunch
  • mRNA
  • Preoptic area

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

Cite this

Competitive PCR for quantitation of gonadotropin-releasing hormone mRNA level in a single micropunch of the rat preoptic area. / Kim, K.; Jarry, H.; Knoke, I.; Seong, Jae Young; Leonhardt, S.; Wuttke, W.

In: Molecular and Cellular Endocrinology, Vol. 97, No. 1-2, 01.11.1993, p. 153-158.

Research output: Contribution to journalArticle

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