RNA extraction and RNA-sequencing method for transcriptomic analysis of Mycobacterium tuberculosis
| Title: | RNA extraction and RNA-sequencing method for transcriptomic analysis of Mycobacterium tuberculosis |
|---|---|
| Authors: | Morgan R. Hiebert, Meenu K. Sharma, Alwyn Go, Christine Bonner, Vanessa Laminman, Morag Graham, Hafid Soualhine |
| Source: | BioTechniques, Vol 77, Iss 1, Pp 23-34 (2025) |
| Publisher Information: | Taylor & Francis Group, 2025. |
| Publication Year: | 2025 |
| Collection: | LCC:Biology (General) |
| Subject Terms: | Mycobacterium tuberculosis, next-generation sequencing, RNA extraction, RNA-sequencing, transcriptomics, Biology (General), QH301-705.5 |
| More Details: | 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. |
| Document Type: | article |
| File Description: | electronic resource |
| Language: | English |
| ISSN: | 07366205 1940-9818 0736-6205 |
| Relation: | https://doaj.org/toc/0736-6205; https://doaj.org/toc/1940-9818 |
| DOI: | 10.1080/07366205.2025.2457887 |
| Access URL: | https://doaj.org/article/8a0cfaa578f54bee9db01dfabac02791 |
| Accession Number: | edsdoj.8a0cfaa578f54bee9db01dfabac02791 |
| Database: | Directory of Open Access Journals |
| ISSN: | 07366205 19409818 |
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| DOI: | 10.1080/07366205.2025.2457887 |
| Published in: | BioTechniques |
| Language: | English |