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Updated: Aug 13, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

3'同位素R物种组成影响miRNA/同位素R变异的可靠量化通过多项A) RT-qPCR:对小RNA-Seq分析验证的影响

Adriana Ferre¹, Lucía Santiago¹, José Francisco Sánchez-Herrero²

¹Human Molecular Genetics Group-Bellvitge Biomedical Research Institute (IDIBELL), 08908 Hospitalet de Llobregat, Spain.

International journal of molecular sciences

|October 28, 2023

概括

使用RT-qPCR验证microRNA (miRNA) 异型是困难的,因为它们的序列相似. 基于多基的RT-qPCR策略显示出交叉反应性,从而损害了对单个miRNA变异的可靠量化.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
生物标志物发现发现

背景情况:

小RNA测序 (小RNA-seq) 识别了微RNA (miRNA) 的异型 (异型R).
通过RT-qPCR验证密切相关的异构基因组,如miR-31-5p变体,由于序列同质性而存在重大挑战.
之前的研究发现了两种miR-31-5p异构Rs在亚精子症中的差异性表达.

研究的目的:

评估三种基于多基A的RT-qPCR策略的区分能力,以区分两个特定的miR-31-5p异构Rs.
在合成和真实生物样本中评估这些RT-qPCR方法的可靠性.

主要方法:

使用了三种不同的基于多基的RT-qPCR策略.
使用合成RNA样本和实际生物标本的测试方法性能.
将RT-qPCR结果与小RNA测序的结果进行比较.

主要成果:

所有测试的基于多基的RT-qPCR策略都显示了两种miR-31-5p异构体之间的显著交叉反应,它们以单个核酸相差.
这种交叉反应性损害了单个miRNA异型的准确量化.
尽管具有交叉反应性,但RT-qPCR结果显示生物样本与小RNA-seq趋势保持一致,因为表达的变化一致.

结论:

基于多基的RT-qPCR策略显示,在高度相似的miRNA异型之间可靠区分的能力有限.
对RT-qPCR验证的miRNA标的仔细选择至关重要,优先考虑具有同质表达模式的正规miRNA以及它们普遍存在的异构Rs.
建议采用这种方法来进行可靠的生物标志物选择和开发临床适用的诊断试验.

关键词:

它是Rs的同位素.的 miRNA 异构形式.聚A) -RT-qPCRR可以使用.有选择性的放大放大.小RNAseq验证策略小RNAseq验证策略

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MicroRNAs

MicroRNAs

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 ends...

RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...