Rose'Meyer Roselyn B, Mellick Albert S, Garnham Bronwyn G, Harrison Glenn J, Massa Helen M, Griffiths Lyn R
School of Health Sciences, Griffith University, GCMC PMB 50, Gold Coast, QLD 9726, Australia.
Brain Res Brain Res Protoc. 2003 Mar;11(1):9-18. 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) 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.
在我们实验室，我们开发了一种定量聚合酶链反应（Q-PCR）策略，用于检测腺苷受体（ADOR）A(1)、A(2A)、A(2B)和A(3)以及雌激素受体（ER）α和β的差异表达。在对受体亚型表达进行SYBR Green I实时分析之前，首先使用脑和子宫的mRNA来优化特异性扩增条件。SYBR Green I提供了一种方便且灵敏的实时检测特异性PCR扩增产物的方法，并能生成标准曲线，据此可确定相对受体丰度。然后进行实时Q-PCR分析，以检测成年雌性Wistar大鼠卵巢切除术后3个月大脑中受体表达水平的变化。与假手术的年龄匹配对照大鼠相比，发现受体水平存在相对和绝对拷贝数的变化。对两种分析方法的评估均将18S rRNA作为大脑中比较基因表达分析的内部参照。本研究结果显示，卵巢切除术后，ADORA(2A)受到优先抑制（下调超过4倍），ADORA(1)、ADORA(3)和ER-β持续下调（超过2倍）。未发现ADORA(2B)或ER-α有变化。本研究中对绝对拷贝数的分析揭示了卵巢切除术后受体表达与大脑中相对受体亚型丰度之间的相关性。
