The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

• Published in: Brain research. Brain research protocols Vol. 11; no. 1; pp. 9 - 18
• Main Authors: Rose’Meyer, Roselyn B, Mellick, Albert S, Garnham, Bronwyn G, Harrison, Glenn J, Massa, Helen M, Griffiths, Lyn R
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2003
• Subjects: Adenosine receptor; Animals; Brain - metabolism; Down-Regulation - genetics; Estrogen receptor; Estrogen Receptor beta; Estrogens - deficiency; Female; Gene Expression Regulation - genetics; Ovariectomy; Polymerase Chain Reaction - methods; Rats; Rats, Wistar; Receptor, Adenosine A2A; Receptor, Adenosine A3; Receptors, Estrogen - genetics; Receptors, Purinergic P1 - genetics; Reverse transcriptase-polymerase chain reaction; RNA, Messenger - analysis; RNA, Messenger - metabolism; RNA, Ribosomal, 18S - analysis; RNA, Ribosomal, 18S - genetics; Neurotransmitter, modulations, transporters and receptors; Estrogen receptor; Reverse transcriptase-polymerase chain reaction; Adenosine receptor; Ovariectomy
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(02)00219-2

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A 1, A 2A, A 2B and A 3, and estrogen receptors (ER) α and β. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA 2A (>4-fold down) and consistent (>2-fold) down-regulation of ADORA 1, ADORA 3, and ER-β, following ovariectomy. No change was found in ADORA 2B or ER-α. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.