改进竞争性RT-PCR法测定缺碘大鼠颅骨中BMP-2基因表达变化

为更加精确地测定碘缺乏状态下大鼠颅骨组织中骨形态发生蛋白 2 (BMP 2 )mRNA表达水平 ,改进竞争性RT PCR方法 ,探讨碘缺乏对骨骼发育影响的分子机制 .将构建的DNA竞争模板克隆入pSportⅠ质粒中 ,利用pSportⅠ质粒中所含有的SP6RNA聚合酶启动子序列 ,将DNA竞争模板在体外转录成为RNA .以此RNA作为竞争性RT PCR中的竞争模板 ,与所提取的总RNA一同进行RT PCR ,消除逆转录效率对定量结果的影响 .定量检测碘缺乏和正常大鼠颅骨总RNA中BMP 2mRNA的水平 .碘缺乏大鼠 (1d～ 84d)颅骨中BMP 2表达含量明显降低 .1d时降低 6 4 3倍 ,以后差距逐渐缩小 ,至 84d相差 1 5 5倍 .结果提示 ,碘缺乏所致的骨骼发育不良与BMP 2表达不足有关 ,结果首次揭示微量元素碘和甲状腺激素与BMP 2基因表达的调控有关     

竞争性RT-PCR测定法及BMP-2mRNA的定量检测

建立竞争性 PCR方法 ,以期用于转录水平基因表达的定量研究 .以检测大鼠颅骨骨细胞总RNA中骨形态发生蛋白 - 2 ( BMP- 2 ,bone morphogenetic protein- 2 ) m RNA含量为例 ,构建大鼠BMP- 2基因的竞争模板 ,以之为内对照进行竞争性 PCR.PCR反应结束后 ,电泳、拍照 ,扫描所扩增条带密度 ,作回归方程 .根据回归方程 ,计算出正常 7d Wistar大鼠颅骨总 RNA中 BMP- 2 m RNA含量为 1 .1 2 5amol/μg RNA.结果显示 ,成功构建了大鼠 BMP- 2基因的竞争模板 ,建立了可以测定大鼠颅骨骨细胞总 RNA中 BMP- 2 m RNA含量的竞争性 PCR方法 .     

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.     

24-Hour variation in gene expression of redox pathway enzymes in rat hypothalamus: effect of melatonin treatment

Abstract

The 24-h changes in medial basal hypothalamic (MBH) gene expression of redox pathway enzymes nitric oxide synthase (NOS)-1 and NOS-2, heme oxygenase (HO)-1 and HO-2, Cu/Zn- and Mn-superoxide dismutases (SOD) and catalase were examined in adult male Wistar rats kept under an alternating regimen of light/dark. Half of the animals received melatonin (～60 μg/day) in the drinking water. After 1 month, rats were killed at six different time intervals, throughout a 24-h cycle. MBH mRNA levels were measured by real-time PCR analysis. In controls, gene expression of NOS-2 and HO-2 peaked at the early light phase while that of HO-1 showed a maximum at the middle of the dark phase. None of MBH mRNAs encoding NOS-1, Cu/Zn-SOD, Mn-SOD and catalase exhibited significant 24-h variations in control rats. Melatonin administration decreased significantly mRNAs for NOS-1, NOS-2, HO-1 and HO-2 as well as changed their 24-h profile. Melatonin augmented gene expression of the antioxidant enzymes Cu/Zn-SOD, Mn-SOD or catalase at certain time intervals only. The results are compatible with the view that the principal indirect (i.e. gene expression of redox pathway enzymes) effect of melatonin on redox pathway in the hypothalamus is mainly exerted via down-regulation of pro-oxidant enzyme mRNAs.