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目的探讨用实时荧光定量PCR检测急性呼吸窘迫综合征(ARDS)患者血清微小RNA(microRNA,miRNA)的表达并选择最优内参照基因。方法用实时荧光定量PCR检测40例ARDS患者及20例体检健康者血清中5个miRNA候选内参照基因miR-16-5p、miR-93-5p、miR-101-3p、miR-191-5p和U6,并用ge Norm法、Norm Finder法、bestkeeper法和相对△Ct法4种算法分析内参照基因稳定性,通过对4种算法稳定值进行排序并计算几何均数的方法分析综合稳定值。结果比较5个候选内参照基因在ARDS患者和体检健康者的表达水平,差异均无统计学意义(P均>0.05)。4种算法综合分析显示,miR-16-5p以最小的稳定值1.32成为综合稳定性最高的内参照基因,其次是miR-101-3p、U6、miR-93-5p和miR-191-5p。结论实时荧光定量PCR法确定ARDS患者血清miRNA最优的内参照基因为miR-16-5p。
Objective To investigate the expression of microRNA (miRNA) in patients with acute respiratory distress syndrome (ARDS) by real-time fluorescence quantitative PCR and to select the optimal internal reference gene. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-16-5p, miR-93-5p, miR-101-3p and miR-191-5p in 5 ARDS patients and 20 healthy controls U6. The stability of internal reference genes was analyzed by ge Norm method, Norm Finder method, bestkeeper method and relative △ Ct method. The stability of the internal reference genes was analyzed by sorting the geometric stability of the four algorithms and calculating the geometric mean. Results There was no significant difference in the expression levels of five candidate internal reference genes among ARDS patients and healthy subjects (P> 0.05). The results of the four algorithms showed that miR-16-5p was the most stable endogenous reference gene with the lowest stable value of 1.32, followed by miR-101-3p, U6, miR-93-5p and miR-191-5p. Conclusion Real-time PCR method to determine the best internal reference miRNA for ARDS patients is miR-16-5p.