Morgan R Hiebert, Meenu K Sharma, Alwyn Go, Christine Bonner, Vanessa Laminman, Morag Graham, Hafid Soualhine
{"title":"RNA extraction and RNA-sequencing method for transcriptomic analysis of <i>Mycobacterium tuberculosis</i>.","authors":"Morgan R Hiebert, Meenu K Sharma, Alwyn Go, Christine Bonner, Vanessa Laminman, Morag Graham, Hafid Soualhine","doi":"10.1080/07366205.2025.2457887","DOIUrl":null,"url":null,"abstract":"<p><p>RNA-sequencing (RNA-seq) technologies have advanced exponentially in recent years, however, the application of RNA-seq to <i>Mycobacterium tuberculosis</i> remains limited. We present a wet-lab and computational protocol for RNA-seq based transcriptomics that was tested on 12 replicates each of 11 clinical isolates of <i>M. tuberculosis</i> (<i>n</i> = 132) grown <i>in vitro</i> with and without pyrazinamide exposure. This RNA extraction method uses low-volume cultures, mechanical lysis, TRIzol<sup>™</sup> phase separation, and column-based purification to produce high yields of pure, intact RNA followed by rRNA depletion and cDNA library preparation. The detection of unique transcripts was optimized at a sequencing depth of 15 million reads. This method detected differential RNA expression in experimental sets with and without pyrazinamide exposure, demonstrating that the method is suitable for RNA-seq applications.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"23-34"},"PeriodicalIF":2.2000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioTechniques","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1080/07366205.2025.2457887","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/2/16 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
RNA-sequencing (RNA-seq) technologies have advanced exponentially in recent years, however, the application of RNA-seq to Mycobacterium tuberculosis remains limited. We present a wet-lab and computational protocol for RNA-seq based transcriptomics that was tested on 12 replicates each of 11 clinical isolates of M. tuberculosis (n = 132) grown in vitro with and without pyrazinamide exposure. This RNA extraction method uses low-volume cultures, mechanical lysis, TRIzol™ phase separation, and column-based purification to produce high yields of pure, intact RNA followed by rRNA depletion and cDNA library preparation. The detection of unique transcripts was optimized at a sequencing depth of 15 million reads. This method detected differential RNA expression in experimental sets with and without pyrazinamide exposure, demonstrating that the method is suitable for RNA-seq applications.
期刊介绍：
BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies.
Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers:
Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions
Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices
Surveys of technical approaches related to broad fields of research
Reviews discussing advancements in techniques and methods related to broad fields of research
Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
