使用体外转录生成功能性miRNA模仿的生产
Teja N Sata1,2, Md Ismail1,2, Amrendra K Sah1
1Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
Current protocols
|June 18, 2025
概括
这项研究提出了一种具有成本效益的体外转录 (IVT) 方法,用于生产微RNA (miRNA) 模仿物. 这些IVT生成的miRNA模拟器在调节基因表达方面,表现出与化学合成基因模拟器相似的疗效.
科学领域:
- 分子生物学分子生物学
- 基因规则 基因规则
- 在RNA治疗方面,RNA疗法.
背景情况:
- 微RNAs (miRNAs) 是基因表达的关键调节者.
- miRNA模拟器是研究miRNA功能和开发治疗方法的重要工具.
- 目前用于miRNA模拟合成的方法包括化学合成和HPLC净化.
研究的目的:
- 开发一种简单,经济,快速的体外转录 (IVT) 方法来生成miRNA模仿.
- 为了比较IVT合成的miRNA效率与化学合成的miRNA效率.
- 为功能性miRNA模拟物提供合成和验证协议.
主要方法:
- 用于体外转录的DNA模板的设计.
- 通过RNA聚合酶驱动的体外转录 (IVT) 来合成miRNA模仿物.
- 基于的柱子净化和RNA链回火.
- 传染miRNA模仿进入哺乳动物细胞.
- 使用qPCR和免疫阻塞验证miRNA模仿表达和功能的验证.
主要成果:
- 为miRNA模仿合成建立了一个高效和快速的IVT协议.
- 通过IVT合成的miRNA仿真体显示出与化学合成的仿真体相比,可比的转录后调节效率.
- 证明了功能miRNA模仿物的转染和验证 (qPCR,免疫阻塞) 的协议.
结论:
- 试管体内转录 (IVT) 提供了一种经济且快速的替代方案,用于生产功能性miRNA模仿物.
- 通过IVT合成的miRNA模仿物可以有效地用于基因表达研究和治疗开发.
- 经过验证的协议促进了miRNA模拟物的合成和功能评估.
相关概念视频
01:22MicroRNAs
3.1K
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
3.1K
02:15Experimental RNAi
6.3K
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
6.3K
01:23RNA Interference
26.5K
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
26.5K
02:30siRNA - Small Interfering RNAs
17.0K
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
17.0K