The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis.
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) alpha and beta. 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-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. 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.
Rose'Meyer
Roselyn B
RB
School of Health Sciences, Griffith University, GCMC PMB 50, Gold Coast, QLD 9726, Australia. r.rosemeyer@mailbox.gu.edu.au
Mellick
Albert S
AS
Garnham
Bronwyn G
BG
Harrison
Glenn J
GJ
Massa
Helen M
HM
Griffiths
Lyn R
LR
D000818:Animals; D001921:Brain / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics*; D047629:Estrogen Receptor beta; D004967:Estrogens / Q000172:deficiency*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D010052:Ovariectomy; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D011960:Receptors, Estrogen / Q000235:genetics*; D018047:Receptors, Purinergic P1 / Q000235:genetics*
D047629:Estrogen Receptor beta; D004967:Estrogens; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D011960:Receptors, Estrogen; D018047:Receptors, Purinergic P1