活动性肺结核患者PBMCs实时定量PCR内参基因的选择

Selection of suitable reference genes for quantitative real-time PCR in PBMCs from active pulmonary tuberculosis patients

目的筛选适用于活动性肺结核患者PBMCs的内参基因。方法用密度梯度离心方法分离活动性肺结核患者PBMCs,然后使用结核分枝杆菌H37Rv裂解液、CFP-10和ESAT-6多肽库、共刺激因子CD28共孵育,或者不添加任何刺激物,培养过夜后收集细胞;使用Trizol提取mRNA,逆转录获得c DNA,然后用6个常用的内参基因ACTB、B2M、DDX5、GAPDH、YWHAZ和18sRNA的特异性引物进行实时定量PCR检测,结果用ge Norm、Norm Finder和Bestkeeper软件进行数据分析。结果 18sRNA基因丰度太高,在PBMCs表达不稳定,DDX5、YWHAZ和B2M的稳定最好,适合作为活动性肺结核患者PBMCs的内参基因,而ACTB和GAPDH稳定性略低。结论筛选出稳定的内参基因DDX5、YWHAZ和B2M,可用于活动性肺结核患者PBMCs基因表达分析。

Objective To screen suitable reference genes of PBMCs for patients with active pulmonary tu- berculosis. Methods PBMCs from patients with active pulmonary tuberculosis were isolated by density gradient centrifugation and then incubated with lysate of Mycobacterium tuberculosis strain H37Rv, peptide pools of CFP-10 and ESAT-6, co-stinmlator CD28 or without any stimulus overnight. The total RNA was isolated using Trizol. Complementary DNA （cDNA） was synthesized using reverse transcription kit. Real-time PCR reactions were performed using primers of 6 reference genes： ACTB, B2M, DDXS, GAPDH, YWHAZ and 18sRNA. The results were analyzed by geNorm, NormFinder and Bestkeeper Software. Results The 18sRNA was abundant and unstable in PBMCs. The expression of DDXS, YWHAZ and B2M was stable, which was suitable reference genes of PBMCs from patients with active pulmonary tuberculosis. The stability of ACTB and GAPDH was slightly lower. Conclusion The stable reference genes DDXS, YWHAZ and B2M are suitable for gene expression analysis of PBMCs in patients with active pulmonary tuberculosis.