RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.
RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.
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DOI：
10.1038/s42003-023-04830-1
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发表时间：
2023-04-24
影响因子：
5.9
通讯作者：
Gu, Lichuan
中科院分区：
文献类型：
--
作者：
Zhang, Fengyu;Wang, Yipeng;Wang, Xiaomeng;Dong, Hongjie;Chen, Min;Du, Ning;Wang, Hongwei;Hu, Wei;Zhang, Kundi;Gu, Lichuan
在过去的30年里，体外转录(IVT)技术已被广泛用于RNA生产或基本转录机制研究。然而，mRNA的定量方法仍需改进。在本研究中，我们设计了一种使用二元荧光猝灭(BFQ)探针和PBCV-1 DNA连接酶的RT-IVT方法，通过荧光共振能量转移(FRET)和RNA-Splinted DNA连接来实时定量mRNA的表达。与现有方法相比，RT-IVT方法成本低廉，无放射性，能实时检测未纯化体系中的mRNA表达，具有较高的灵敏度和选择性。用该方法对T7RNA聚合酶和大肠杆菌RNA聚合酶的全酶活性进行了鉴定。然后，我们使用带有不同颜色荧光团的BFQ探针，在RT-PCR热循环仪上对三个T7启动子的实时mRNA进行多路定量，每个靶标都有不同的荧光载体。最终，我们创建了一种廉价的多重方法来实时量化mRNA的生产，未来的研究可以使用这些方法来测量转录抑制物与其目标DNA序列的亲和力。一种新的方法(RT-IVT)利用FRET中的荧光猝灭剂&DNA连接酶和RNA拼板DNA连接方法来实现廉价、非放射性和选择性的mRNA生产的定量。
For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence. A new method (RT-IVT) leverages fluorescence quenchers & DNA ligase in FRET and RNA-splinted DNA ligation methods to allow inexpensive, nonradioactive and selective mRNA production quantification.
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影响因子：
46.9
作者：
Mitchell, P
通讯作者：
Mitchell, P
DOI：
10.1073/pnas.2233244100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Bratu, DP;Cha, BJ;Tyagi, S
通讯作者：
Tyagi, S
DOI：
10.1007/978-1-4939-2425-7_35
发表时间：
2015-01-01
期刊：
PROTEIN-PROTEIN INTERACTIONS: METHODS AND APPLICATIONS, 2ND EDITION
影响因子：
--
作者：
Du, Yuhong
通讯作者：
Du, Yuhong
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
DOI：
10.1073/pnas.95.20.11538
发表时间：
1998-09-29
影响因子：
11.1
作者：
Sokol, DL;Zhang, XL;Gewitz, AM
通讯作者：
Gewitz, AM