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Purification of Bacterial RNA from Infected Macrophages

感染巨噬细胞的细菌RNA的纯化

LL Lior Lobel
NS Nadejda Sigal
AP Anna Pasechnek
AH Anat A. Herskovits

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1660 浏览次数: 11407

评审: Fanglian HeYoko Eguchi

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Cover of Molecular Microbiology, featuring study using the protocol.

Feb 2015

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Abstract

Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).

Keywords: Listeria monocytogenes (单核细胞增生李斯特氏菌) search Bacterial RNA extraction (细菌RNA提取) search Intracellular bacterial RNA (细胞内的细菌RNA) search

Materials and Reagents

  1. Cell scrapers (Thermo Fisher Scientific, NuncTM, catalog number: 179693 )
  2. MF-Millipore filters (Merck Millipore Corporation, catalog number: HAWP04700 )
  3. Cell culture dishes (Greiner Bio One International GmbH, catalog number: 639160 )
  4. Eppendorf tubes (Corning Inc., Axygen, catalog number: MCT175C )
  5. Pipettes sterile (25 ml) (Greiner Bio One International GmbH)
  6. Falcon tubes (50 ml) (Greiner Bio One International GmbH)
  7. Listeria monocytogenes 10403S (Daniel Portnoy’s lab stock) (Becavin et al., 2014)
  8. Bone marrow (from C57B/6 female mice; ordered from Harlan labs Israel)
  9. Liquid nitrogen
  10. RNase-free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  11. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
  12. L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  13. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360088 )
  14. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  15. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Gentamicin (Sigma-Aldrich, catalog number: G1397 )
  17. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  18. Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
  19. Brain Heart Infusion (BHI) (Merck Millipore Corporation, catalog number: 1104930500 )
  20. Phenol saturated (pH 4.3) (Thermo Fisher Scientific, catalog number: BP1751I-400 )
  21. Chloroform (Thermo Fisher Scientific, catalog number: BP1145-1 )
  22. Isoamyl alcohol (Sigma-Aldrich, catalog number: W205702 )
  23. Sodium acetate Anhydrous (Sigma-Aldrich, catalog number: W302406 )
  24. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
  25. DNase I, RNase-free (supplied with MnCl2) (1 U/µl) (Fermentas, catalog number: EN0521 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: EN0521”.
  26. 10% Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4522 )
  27. Ethanol absolute (Merck Millipore Corporation, catalog number: 1070174000 )
  28. M-CSF (L-929 conditioned medium) (Englen et al., 1995)
    Note: Alternatively M-CSF can be bought from Sigma (Sigma-Aldrich, catalog number: SRP3221 ).
  29. BMDM + PS media (filter sterilized) (see Recipes)
  30. BMDM no PS media (filter sterilized) (see Recipes)
  31. AE buffer (see Recipes)
  32. Phenol-chloroform-IAA (see Recipes)
  33. Chloroform-IAA (see Recipes)

Equipment

  1. CO2 forced-air incubator (Thermo Fisher Scientific, model: 3111 )
  2. Kontes glass holder (Thermo Fisher Scientific, catalog number: K953755-0045 )
  3. Speed-Vac Concentrator (Thermo Fisher Scientific, catalog number: SPD131DDA )
  4. Vortex-Genie 2 (Scientific Industries, model: G560E )
  5. Nanodrop 1000 (Thermo Fisher Scientific)
  6. 30 °C incubator (Thermo Fisher Scientific)
  7. 65 °C heat block (Thermo Fisher Scientific)
  8. 4 °C table centrifuge (Thermo Fisher Scientific, EppendorfTM, model: 5417R )

Procedure

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文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lobel, L., Sigal, N., Pasechnek, A. and Herskovits, A. A. (2015). Purification of Bacterial RNA from Infected Macrophages. Bio-protocol 5(22): e1660. DOI: 10.21769/BioProtoc.1660.

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分类

微生物学 > 微生物遗传学 > RNA > RNA 提取

微生物学 > 微生物-宿主相互作用 > 体外实验模型 > 细胞系

分子生物学 > RNA > RNA 提取

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