Journal List > Ann Clin Microbiol > v.19(1) > 1078550

Ann Clin Microbiol. 2016 Mar;19(1):20-23. English.
Published online March 24, 2016.  https://doi.org/10.5145/ACM.2016.19.1.20
Copyright © 2016 The Korean Society of Clinical Microbiology
An Effective Method of RNA Extraction from Mycobacterium tuberculosis
Tae Sang Oh,1 Hee Yoon Kang,2 You Sun Nam,1 Young Jin Kim,2 Eun Kyung You,2 Min Young Lee,2 Sun Young Cho,2 and Hee Joo Lee2
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, Email: leehejo@khmc.or.kr
Received October 26, 2015; Revised December 30, 2015; Accepted February 25, 2016.

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 renders the cells resistant to lysis. Using TRIzol reagent and several powerful bead-beating steps, a high quantity of RNA was obtained.

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


Tuberculosis due to infection with Mycobacterium tuberculosis is one of the most important communicable diseases [1]. Because M. tuberculosis grows more slowly than most other bacteria, direct analysis of RNA expression is a major area of interest [2]. To successfully quantify the expression of specific M. tuberculosis genes by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) or RNA sequencing (RNA-Seq), it is important to use intact and pure RNA that is free of DNA and proteins as a template [3]. However, RNA extraction from M. tuberculosis is problematic due to its thick, waxy cell wall rich in mycolic acid, which renders the cells resistant to lysis [4]. Furthermore, because RNA is subject to degradation during any extraction or purification steps [5], sophisticated handling techniques under optimal conditions are necessary.

Various methods for extraction of mycobacterial RNA have been reported based on enzymatic hydrolysis, chemical treatment, french pressure cell rupture, bead-beating or sonication [2, 4]. We first tried to extract RNA based on enzymatic hydrolysis with lysozyme and RNeasy Protect Bacteria Mini Kit (Qiagen, Venlo, Netherlands) for purify RNA but it was problematic. Larsen et al. introduced two methods for extraction of RNA from M. tuberculosis: the RNA-TRIzol protocol and the Fast Prep method [6]. Each protocol of Larsen et al. used TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) or bead beater individually. We modified the above methods for our laboratory and successfully extracted intact and pure RNA. Here, we describe our simple RNA extraction method that involves simultaneous use of TRIzol reagent (Thermo Fisher Scientific) and a bead beater in a single extraction step (Table 1). To avoid RNA degradation, samples should be handled quickly and be kept on ice at all times, except during reactions with reagents.


Table 1
Flow of RNA extraction of M. tuberculosis
Click for larger image

Fifteen multidrug-resistant M. tuberculosis strains, fifteen extensively drug-resistant M. tuberculosis strains, and M. tuberculosis H37Rv were cultured in 30 mL Middlebrook 7H9 broth (BD, Franklin Lakes, NJ, USA) supplemented with 10% oleic acid, albumin, dextrose, catalase enrichment (BD), and 0.5% (v/v) glycerol in a 37℃ incubator for 2-3 weeks. An aliquot (7 mL) of culture was then transferred to 15 mL screw-cap tubes and centrifuged at 2,200 g and 4℃ for 10 min with removal of supernatant to harvest mycobacterial cells, which were washed in 1 mL Tris-EDTA buffer (pH 7.5) by tapping tube to remove excess 7H9 broth, centrifuged again using identical conditions, then resuspended in 1 mL RNAprotect Bacteria Reagent (Qiagen) by vortex-mixing. The cells were then incubated for 5 min at room temperature and centrifuged again. After removal of the supernatant, the pellet can be preserved for several months at –80℃ or proceed to the next step. The pellet was resuspended in 1 mL TRIzol reagent (Thermo Fisher Scientific), transferred to a Lysing Matrix B tube (MP Biomedicals, Santa Ana, CA, USA) containing 0.1 mm silica beads, and processed four times using a Fast Prep-24 5G (MP Biomedicals) instrument for 45 s using a speed setting of 6.5. After each bead-beating run, the tube was incubated in an ice block, and at room temperature in the last run. Chloroform (0.2 mL) was added to lysates and tubes were shaken manually for 30 s. Lysates were incubated for 5 min at 4℃ and centrifuged at 13,400 g and 4℃ for 15 min. The aqueous upper layer of chloroform containing RNA was transferred to a new 1.5 mL tube and RNA was precipitated by adding 0.5 mL isopropyl alcohol, followed by incubation for 20 min at –80℃. The precipitated RNA was centrifuged at 13,400 g and 4℃ for 10 min and the supernatant was decanted. The RNA pellet was washed by brief vortexing, mixing with 1 mL 75% ethanol, re-centrifuged at 13,400 g and 4℃ for 5 min, air-dried briefly, and dissolved in 50 µL diethylpyrocarbonate (DEPC)-treated water. RNA concentration and purity (A260/A280 nm) were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and electrophoresis in a 1% agarose gel followed by ethidium bromide staining. To eliminate genomic DNA contamination, DNA was digested using a TURBO DNA-free Kit (Thermo Fisher Scientific). RNA was diluted with DEPC-treated water to 250 ng/µL and treated with 6 U of DNase (the maximum recommended by the manufacturer) at 37℃ for 1 h. The measured concentration of extracted RNA treated with DNase were ~126-208 ng/µL and purity (A260/A280 nm) of that were 1.66-1.84, respectively. Two clear bands of 16S and 23S rRNA were identified on the gel (Fig. 1). PCR amplification of rpoB (housekeeping gene) was performed to check for genomic DNA contamination. The forward and reverse primers used were newly designed by Bioneer (Daejeon, Korea), and their sequences are as follows: 5'-GTTCAAGG TGCTGCTCAAAG-3'and 5'-GGACAGATTGATTCCCAGGT-3', respectively. The annealing temperature was 59℃. Then, amplified products were validated free of genomic DNA by electrophoresis in a 2% agarose gel and ethidium bromide staining (Fig. 1).


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.
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For RNA extraction, we compared two M. tuberculosis growth media: Enriched Middlebrook 7H9 broth and Mycobacteria Growth Indicator Tube (MGIT) (BD). Seven milliliters of bacterial culture were used to extract total RNA using the extraction method described above. However, RNA extracted from MGIT cultures showed low yield or degradation (Fig. 2), in contrast to previous report [7]. Two culture media are almost same except casein peptone and fluorescence indicator. However, we inoculated a loopful of bacteria in media directly, the rate of proliferation could be faster than usual. The volume (7 mL) and thin tube of MGIT could be insufficient for the growth of bacteria for 3 weeks. Because the volume and container of 7H9 broth we used were 30 mL and Erlenmeyer flask, environment for growth of mycobacteria could be more ideal than that of MGIT. Also, we investigated whether the diameter of the beads used for bacterial cell lysis could affect the yield and/or quality of RNA. We extracted RNA from M. tuberculosis using beads of diameters 0.1 mm, 0.2 mm and 0.2-0.5 mm mixture; the results did not differ significantly among the three bead diameters. In the early stage of this study, RNA was purified using an RNeasy Protect Bacteria Mini Kit (Qiagen); however, this led to a loss of over 50% of total RNA after cleanup. It is believed that the RNA could not adhere to the surface of the column during purification. To reduce RNA loss, we performed DNase treatment after precipitation of RNA without column cleanup; this reduced RNA loss to ~20%. Thus, if genomic DNA is not identified by gel electrophoresis and the RNA is of reasonable purity, additional purification is unnecessary.


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.
Click for larger image

Extraction of intact and pure RNA is important but problematic. Using TRIzol reagent and several powerful bead-beating steps, a high quantity of RNA was obtained. After treatment with a high concentration of DNase, the extracted RNA is available for use in subsequent experiments.

ACKNOWLEDGMENTS

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A2004931).

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;25:675–676.
3. Fleige S. RNA integrity and the effect on the realtime qRT-PCR performance. Mol Aspects Med 2006;27:126–139.
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.
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.
6. Larsen MH, Biermann K, Tandberg S, Hsu T, Jacobs WR Jr. Genetic manipulation of Mycobacterium tuberculosis. Curr Protoc Microbiol 2007;Chapter 10:Unit 10A.2.
7. Rodrigues L, Machado D, Couto I, Amaral L, Viveiros M. Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex. Infect Genet Evol 2012;12:695–700.