感染巨噬细胞的细菌RNA的纯化

Lior Lobel; Nadejda Sigal; Anna Pasechnek; Anat A. Herskovits

doi:10.21769/BioProtoc.1660

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1660 浏览次数: 10243

评审: Fanglian HeYoko Eguchi

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Feb 2015

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

γ-H2AX作为标志物采用细胞内蛋白质印迹法（ICW）定量HeLa细胞中Colibactin相关基因毒性

Sophie Tronnet and Eric Oswald

2018年03月20日 7213 阅读

肠凝聚型大肠埃希杆菌对HEK293细胞的粘附测定

Jorge Luis Ayala-Lujan and Fernando Ruiz-Perez

2018年04月20日 5940 阅读

研究酗酒酒精毒性对小鼠肺泡巨噬细胞影响的细胞内侵袭和杀伤试验

Victor Jimenez Jr and Fernando P Monroy

2019年01月20日 5715 阅读

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)

Bacterial RNA extraction (细菌RNA提取)

Intracellular bacterial RNA (细胞内的细菌RNA)

Materials and Reagents

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

Equipment

CO₂ forced-air incubator (Thermo Fisher Scientific, model: 3111 )
Kontes glass holder (Thermo Fisher Scientific, catalog number: K953755-0045 )
Speed-Vac Concentrator (Thermo Fisher Scientific, catalog number: SPD131DDA )
Vortex-Genie 2 (Scientific Industries, model: G560E )
Nanodrop 1000 (Thermo Fisher Scientific)
30 °C incubator (Thermo Fisher Scientific)
65 °C heat block (Thermo Fisher Scientific)
4 °C table centrifuge (Thermo Fisher Scientific, Eppendorf^TM, model: 5417R )

Procedure

English

中文翻译

文章信息

版权信息

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.