Rapid and direct microRNA quantification by an enzymatic luminescence assay.
Rapid and direct microRNA quantification by an enzymatic luminescence assay.
复制标题
DOI：
10.1016/j.ab.2012.06.021
复制
发表时间：
2012-10-01
影响因子：
2.9
通讯作者：
Golovlev V
中科院分区：
文献类型：
--
作者：
Sun Y;Gregory KJ;Chen NG;Golovlev V
建立了一种快速、灵敏的microRNA（miRNA）表达定量分析方法。该测定法直接利用miRNA作为引物结合环状单链DNA模板，然后进行滚环扩增。在DNA聚合和扩增过程中释放的无机焦磷酸盐（PPi）分子的检测是通过多酶系统进行的。PPi通过ATP-硫酸化酶转化为ATP，ATP-硫酸化酶为荧光素酶提供能量以氧化胰蛋白酶并产生光。实验结果表明，该方法具有超过三个数量级的动态范围，并能够区分具有高同源性序列的miRNA。对人心脏组织中9种miRNA的定量表明，该测定法与TaqMan真实的时间PCR测定法之间具有高度的跨平台一致性，R2 = 0.941。该检测需要更少的试剂，可以在等温条件下进行而无需热循环，并且能够在不到一小时的时间内检测miRNA。与真实的实时PCR和基于微阵列的检测方法相比，该方法为生命科学研究、药物发现和临床诊断中的miRNA定量提供了更简单、更快速、更便宜的平台。
A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay utilizes miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated a high cross-platform consistency between this assay and the TaqMan real time PCR assay with R2 = 0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than an hour. Compared to the real time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.
登录
查看更多内容
影响因子：
64.8
作者：
Lu, J;Getz, G;Golub, TR
通讯作者：
Golub, TR
影响因子：
56.9
作者：
Lagos-Quintana, M;Rauhut, R;Tuschl, T
通讯作者：
Tuschl, T
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
12.3
作者：
Loscher CJ;Hokamp K;Kenna PF;Ivens AC;Humphries P;Palfi A;Farrar GJ
通讯作者：
Farrar GJ
影响因子：
6.4
作者：
Nelson, Peter T.;Wang, Wang-Xia;Rajeev, Bernard W.
通讯作者：
Rajeev, Bernard W.