低氧性肺动脉高压大鼠肺动脉平滑肌RT-PCR检测中内参基因的选择

Selection of suitable reference genes for normalization of RT-PCR in rat pulmonary artery smooth muscle cells of hypoxia pulmonary artery hypertension.

目的评价实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达时12个候选内参基因表达的稳定性,获得最适合的内参基因。方法以低氧性肺动脉高压大鼠肺动脉平滑肌为研究对象,选择文献报道的常用12种内参基因为候选内参基因,利用ge Norm和Norm Finder程序分析实时荧光定量PCR数据,筛选出最适内参基因。结果 12个候选内参基因在低氧性肺动脉高压大鼠肺动脉平滑肌表达稳定性由强到弱顺序为:TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1,平均表达稳定度(M值)均<0.5,ge Norm和Norm Finder评估后推荐使用TBP和B2M一起作为该研究时的内参基因。结论同时使用TBP和B2M是实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达的最适合内参基因,为低氧性肺动脉高压相关基因研究提供最优内参基因。

Objective To compare and select the suitable reference genes in real-time quantitative PCR analysis of rat pulmonary artery smooth muscle cells mRNA expression level of pulmonary hypertension. Methods To choose appropriate reference gene, the expression of twelve commonly use housekeeping genes were examined in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension by using geNorm and NormFinder programs. Results The expression consis- tency of 12 genes was （from high to low） ： TBP 〉 B2M 〉 HPRT1 〉 HMBS 〉 RPL13a 〉 18sRNA 〉 PPIA 〉 ACTB 〉 GUSB 〉 TFRC 〉 GAPDH 〉 PGK1. The average expression stability （M） values of them were low than 0. 5. TBP and B2M reference genes were recommended to use in the same condition. Conclusion TBP and B2M reference genes were the most suitable combination of the reference genes for real-time quantitative PCR analysis in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension.