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Oh, Kang, Nam, and Kim: An Effective Method of RNA Extraction from Mycobacterium tuberculosis
Original Article

Ann Clin Microbiol 2016;19(1):20-23.

Published online: 12 March 2016

DOI: https://doi.org/10.5145/ACM.2021.19.1.20

An Effective Method of RNA Extraction from Mycobacterium tuberculosis

Tae Sang Oh, Hee Yoon Kang, You Sun Nam, Young Jin Kim2,

1Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, Korea

2Department of Laboratory Medicine, Kyung Hee University School of Medicine, Seoul, Korea

Correspondence: Hee Joo Lee, Department of Laboratory Medicine, Kyung Hee University School of Medicine, 23, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea. (Tel) 82-2-958-8672, (Fax) 82-2-958-8609, (E-mail) leehejo@khmc.or.kr

Received 26 October 2015       Revised 30 December 2015       Accepted 25 February 2015

Copyright © 2016 The Korean Society of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In the RNA-based study, it is important to extract high-quality RNA. However, RNA extraction from Mycobacterium tuberculosis is problematic due to its thick, waxy cell wall rich in mycolic acid, which ren-ders the cells resistant to lysis. Using TRIzol reagent and several powerful bead-beating steps, a high qua-ntity of RNA was obtained.

Keywords: Mycobacterium tuberculosis, RNA extraction, Quantitative reverse transcription polymerase chain reaction

References

1. World Health Organization. Global tuberculosis report 2014. World Health Organization;2014.
2. Mangan JA, Sole KM, Mitchison DA, Butcher PD. An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria. Nucleic Acids Res. 1997; 25:675–6.
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3. Fleige S and Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27:126–39.
4. Hia F, Chionh YH, Pang YL, DeMott MS, McBee ME, Dedon PC. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides. Nucleic Acids Res. 2015; 43:e32.
crossref
5. Pérez-Novo CA, Claeys C, Speleman F, Van Cauwenberge P, Bachert C, Vandesompele J. Impact of RNA quality on reference gene expression stability. Biotechniques. 2005; 39:52. 54, 56.
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Fig. 1.
Ethidium bromide-stained 1% agarose gel of RNA extracted from clinical isolates of M. tuberculosis. All lanes showed 16S and 23S rRNA bands. Lanes 1 to 33: M. tuberculosis clinical isolates; lanes 25, 26 and 27 were identical isolates with lanes 19, 23 and 24 respectively.
acm-19-20f1.tif
Fig. 2.
Ethidium bromide-stained 1% agarose gel of RNA extracted from M. tuberculosis clinical isolates and strain H37Rv. Lanes 2 to 5: 16S and 23S rRNA bands. Lane 1: M. tuberculosis clinical isolate grown in MGIT; Lane 2: M. tuberculosis H37Rv cultured in Enriched Middlebrook 7H9 Broth; Lanes 3 to 5: M. tuberculosis clinical isolates cultured in Enriched Middlebrook 7H9 Broth; Lanes 6 to 7: M. tuberculosis clinical isolates cultured in MGIT.
acm-19-20f2.tif
Table 1.
Flow of RNA extraction of M. tuberculosis
Phase Time
Sample homogenizing  
Cell harvest 35 min
Spin (7 mL of bacterial cells) 10 min
Decant of supernatant and wash with Tris buffer, Spin 10 min
Decant of supernatant and incubation with RNA protect bacteria reagent 5 min
Spin 10 min
Cell disruption 43.5 min
Bead beating with Trizol solution and incubation (repeat four times) 23 min
Shaking with chloroform 0.5 min
Incubation 5 min
Spin 15 min
RNA isolation  
RNA precipitation 25 min
RNA precipitation with isopropyl alcohol 20 min
Spin 5 min
RNA wash and resuspension 5 min
Decant of supernatant and wash with ethyl alcohol Briefly
Spin 5 min
Air dry Briefly
Dissolve with DEPC-treated water  
DNA digestion  
Activation of DNase 60 min
Incubation with TURBO DNase buffer and TURBO DNase 60 min
Inactivation of DNase 6.5 min
Incubation with DNase inactivation reagent 5 min
Spin and transfer RNA of supernatant to new tube 1.5 min
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