Full Length Research Paper

Quantification of substance P mRNA expression in the midbrain of ovariectomized migraine rats with SYBR green I real-time polymerase chain reaction

Ting-min Yu*, Gang Yao, Yu-hong Man and Xi-jing Mao

Department of Neurology, the Second Hospital of Jilin University, Changchun, Jilin 130041, China.

*Corresponding author. E-mail: yecolor@163.com. Tel: +86-431-88796298.

Abbreviations: RT-PCR, Real-time polymerase chain reaction; SP, substance P; PAG, periaqueductal gray; Ct, threshold cycles.

Accepted 8 July, 2010

   Abstract

This study was designed to develop a SYBR green I-based real-time polymerase chain reaction (RT-PCR) for quantitative detection of substance P (SP) mRNA in the midbrain of ovariectomized migraine rats and to evaluate the effects of estradiol on the mRNA expression of SP in order to shed light on the mechanisms underlying the pathogenesis of migraine and estrogen-conferred protection against migraine. 24 female rats were randomly assigned to the following groups: A: non-migraine controls; B: migraines; C, migraine rats receiving low estradiol; D, migraine rats receiving high estradiol. One week following ovariectomy, migraine was induced in groups B, C and D by nitroglycerin (i.p.). Behavior changes before and after migraine was examined. A SYBR green I-based RT-PCR assay was established to measure the absolute levels of SP mRNA in the midbrain. Behavioral changes in group D were significantly mitigated when compared with those in group B, whereas no marked behavioral changes were noted in groups C and B. In addition, mRNA copies of SP in group B were remarkably lower than group A, while the level of SP mRNA in both groups C and D was higher than group B, although no significance was reached (P > 0.05). SP mRNA expression decreased in the midbrain of migraine rats when compared with the non-migraine controls. High doses of estrogen partially restored SP expression in migraine rats and reduce migraine attack. Our study validated the SYBR green I-based RT-PCR technique for quantitative detection of SP mRNA.

Key words: Substance P, migraine, estrogen, midbrain, real-time quantitative polymerase chain reaction, rats.