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- 2017
U6和let-7a作为定量检测miRNAs的内参在大鼠软骨中稳定性的比较
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Abstract:
摘要：目的 比较U6和let-7a两种内参在RT-qPCR方法检测大鼠股骨头软骨样本microRNAs(miRNAs)的稳定性。方法 取软骨生成过程的新生(D0)、断乳(D21)、性成熟(D42)3个时间节点的雌性近交系DA大鼠股骨头软骨，提取总RNA，用RT-qPCR方法分别在U6和let-7a两种内参体系下，检测miR-1、-25、-26a、-140、-146a、-150、-181a、-195、-223、-337等的表达，并与课题组前期获得的二代测序绝对定量结果进行比较，综合评价两种内参的稳定性。结果 在D42样本中 U6(P=0.045)、let-7a(P=0.0215)的相对值出现了显著性差异；在两种内参体系中miR-26a、-140、-223、-337的表达趋势相同，miR-1、-146a差异无统计学意义，miR-25、-150、-181a、-195都出现了显著性差异(P<0.05)。综合二代测序绝对定量结果比较，let-7a稳定性优于U6。结论 let-7a稳定性优于U6，更适合做软骨miRNA定量的内参。
ABSTRACT: Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RT-qPCR by using femoral head samples of cartilage tissue from inbred DA rats. Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points, i.e. at birth (D0), ablactation (D21) and maturation (D42). The expressions of different miRNAs (miR-1, -25, -26a, -140, -146a, -150, -181a, -195, -223 and -337) were detected by RT-qPCR using U6 or let-7a as the internal reference. The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references. Results The relative values of U6 (P=0.045) and let-7a (P=0.0215) revealed significant difference in the D42 sample. Both in U6 and let-7a systems, miR -26a, -140, -223, and -337 showed a similar tendency in expression and quantification but miR-1 and -146a did not have significant differences. miR -25, -150, -181a and -195 differed significantly (P<0.05). Comparison of absolute quantification results between the two generations’ sequencing showed that let-7a is more stable than U6. Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue
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