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Purification of Bacterial RNA from Infected Macrophages
感染巨噬细胞的细菌RNA的纯化   

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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), Bacterial RNA extraction(细菌RNA提取), Intracellular bacterial RNA(细胞内的细菌RNA)

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

  1. Infection
    Day 1:
    1. Seed 2.0 x 107 bone marrow derived macrophages (BMDM) in 145 mm dishes with 30 ml BMDM + PS media, 3 plates for each bacterial strain being analyzed.
    2. Pick a bacterial colony from a BHI plate and inoculate into 10 ml of BHI medium (repeat for each sample), incubate at 30 °C incubator, slanted without shaking overnight.
    Day 2:
    1. Pre-warm BMDM no PS medium and PBS at 37 °C.
    2. Wash macrophage monolayer twice with 25 ml of warmed PBS to remove the antibiotics.
    3. Add 30 ml fresh medium.
    4. Wash 1.5 ml of overnight bacterial cultures twice with PBS.
    5. Infect macrophages at MOI of 90:1 (in favor for the bacteria) for wild-type L. monocytogenes 10403S. When mutants defected in intracellular growth are used, consider increasing the MOI.
    6. Infect each plate 15 min apart (same as in step A5).
    7. Following 0.5 h incubation (in respect to each sample infection time), wash monolayers twice with 30 ml PBS. Add 30 ml pre-warmed BMDM no PS medium.
    8. At 1 h post infection, add 30 µl of gentamicin (1:1,000) to kill extracellular bacteria.
    9. Prepare filter apparatus and ice-cold RNase-free water.
    10. At 6 h post infection, harvest the bacteria. Treat each plate individually.
      1. Wash monolayers with PBS once.
      2. Add 20 ml of ice-cold RNase-free water.
      3. Using a cell scraper, scrape and lyse cells by pushing the liquid in front of the scraper.
      4. Collect by pipetting the lysed monolayer in a 50ml conical tube. Vortex for 30 sec. Centrifuge 2,000 rpm (720 x g) /3 min/ 4 °C.
      5. Pass supernatant through filter apparatus. Using tweezers, put the filter membrane in 15 ml conical tubes, loosely rolled.
      6. Immediately freeze filters by dipping the 15 ml tubes in liquid nitrogen.
      7. Prepare the filter apparatus for the next sample and repeat.

  2. Nucleic acids extraction
    Day 3:
    1. Prepare 1:1 mix of acidified phenol:chloroform, 400 µl for each sample. Mix in a separate tube, and then expel the resulting aqueous layer aspirated off.
    2. Thaw your filter-containing tubes on ice. Insert each tube fully into ice.
    3. To each filter-containing tube add 650 µl AE buffer. Add buffer to all tubes.
    4. Working quickly, vigorously vortex your filter containing tube so that the filter whisks to the periphery of the tube and the buffer fully washes over the filter. It may be necessary to additionally vortex the tube while inverted to fully wash the bacteria off the filter. Always keep tube cold by placing it back on ice. Repeat for all tubes. It may be useful to spin-down the suspension shortly (1 min/750 rpm/60 x g) at the end of the process.
    5. Transfer the bacteria-containing buffer to the 1.5 ml Eppendorf tube containing 40 µl of 10% SDS and 400 µl of phenol/chloroform mix. Repeat for all tubes. It may be necessary to spin down tubes shortly (1 min/750 rpm) to get residual buffer from the filter.
    6. Place all of your tubes in the multi-tube vortex device, and vortex at full speed for 10 min.
    7. Incubate the tubes at 65 °C heat block for 10 min.
    8. Centrifuge at maximum speed (14,000 RPM / 20,817 x g) for 5 min.
    9. Transfer the aqueous layer (about 400 µl) from each tube to the 1.5 ml tube containing 40 µl 3 M sodium acetate (pH 5.2) and 1.0 ml 100% ethanol. Vortex each tube thoroughly.
    10. Incubate your sample at -80 °C 1 h.
    11. Centrifuge at 4 °C for 20 min at maximum speed (14,000 RPM / 20,817 x g).
    12. Carefully aspirate off the ethanol from each tube. Add 500 µl cold 70% ethanol to each sample and vortex thoroughly.
    13. Centrifuge at 4 °C for 20 min at maximum speed.
    14. Carefully aspirate off the ethanol from each tube. Dry your samples for approximately 2 min in a speed-Vac device without heating. It is critical not to over-dry your samples.
    15. To each sample, add 25 µl RNase-free water. Incubate at room temperature for 20 min. Carefully vortex and spin-down.
    16. Analyze the RNA concentration in the samples using the NanoDrop. Expect to extract about 0.5-1 µg/plate.
    17. You can unite the technical replicates at this stage.

  3. DNase treatment
    1. Set reaction:
      RNA
      up to 2 µg (max 44 µl)
      10x DNase buffer
      5 µl
      H2O
      complete to 50 µl
      DNase
      1 µl
      Total
      50 µl
    2. Incubate at 37 °C for 45 min.
    3. Add 450 µl RNase-free water and 500 µl of Phenol-Chloroform-IAA mix, separate phases by 2 min centrifugation at maximal speed.
    4. Transfer the aqueous layer to a new tube and add 500 µl chloroform-IAA mix, vortex and separate phases by 2 min centrifugation at maximal speed.
    5. Transfer the aqueous layer to a new tube and add 1 ml of ethanol and 50 µl of 3 M sodium acetate (pH 5.2). Vortex.
    6. Incubate for 1 h at -80 °C.
    7. Centrifuge at 4 °C for 20 min at maximal speed.
    8. Carefully aspirate off the ethanol from each tube. Add 500 µl cold 75% ethanol to each sample and vortex thoroughly.
    9. Centrifuge at 4 °C for 20 min at maximum speed.
    10. Carefully aspirate off the ethanol from each tube. In a speed-Vac without heating, dry your samples for approximately 2 min. It is critical not to over-dry your samples.
    11. Add 12 µl of RNase-free water, incubate for 2 min at room temperature, vortex, and spin-down. Keep the RNA on ice.
    12. Analyze the RNA concentration in the samples using the NanoDrop. Expect to extract about 100 ng/sample.

Representative data

We provide representative data of induction of the virulence gene hly of L. monocytogenes during intracellular growth in macrophages compared to growth in BHI medium (Figure 1). mRNA was purified from intracellular bacteria 6 hours post infection at M.O.I of 90:1 and from bacteria grown to mid log (OD600 = 0.5) in BHI medium.


Figure 1. Transcription of hly during growth of L. monocytogenes in BMDMs (6 h.p.i.) and BHI lab medium. Error bars represent 95% confidence interval. Results are representative of 3 repeats (N=3). RQ: Relative quantity

Notes

  1. Check the morphology of the infected cells before and after infection. The cells should be viable during the infection process.
  2. Be very careful when handling the membranes-use tweezers and touch only the edges.
  3. When passing the samples through the filter apparatus, first filter RNase-free water to verify that the vacuum is working and that there is no blockage in the system.
  4. You should observe a reduction in nucleic acid concentration following DNase I treatment. If you don’t observe a reduction, consider another round of DNase I treatment.

Recipes

  1. BMDM+PS media (filter sterilized)
    DMEM
    250 ml
    FBS (inactivated 30 min at 54 °C)
    100 ml
    M-CSF (L-929 conditioned medium) (Englen et al., 1995)
    150 ml
    Glutamine
    5 ml
    Sodium pyruvate
    5 ml
    β-Mercaptoethanol
    0.5 ml
    Pen/Strep
    5 ml
  2. BMDM no PS media (filter sterilized)
    DMEM
    255 ml
    FBS (inactivated 30 min at 54 °C)
    100 ml
    M-CSF (L-929 conditioned medium) (Englen et al., 1995)
    150 ml
    Glutamine
    5 ml
    Sodium pyruvate
    5 ml
    β-Mercaptoethanol
    0.5 ml
  3. AE buffer
    50 mM NaOAc (pH 5.2)
    10 mM EDTA
    RNase-free water
  4. Phenol-chloroform-IAA
    Phenol 25 ml
    Chloroform 24 ml
    Isoamyl alcohol 1 ml
  5. Chloroform-IAA
    Chloroform 24 ml
    Isoamyl alcohol 1 ml

Acknowledgments

This protocol was adapted from the previously published studies (Lobel et al., 2015; Kaplan Zeevi et al., 2013; Lobel et al., 2012). This work was supported by the Israel Science Foundation and the ERA-NET PathoGenoMics (funded by MOH Israel) grants to AAH. L.L is supported by the Argentinian friends of TAU.

References

  1. Becavin, C., Bouchier, C., Lechat, P., Archambaud, C., Creno, S., Gouin, E., Wu, Z., Kuhbacher, A., Brisse, S., Pucciarelli, M. G., Garcia-del Portillo, F., Hain, T., Portnoy, D. A., Chakraborty, T., Lecuit, M., Pizarro-Cerda, J., Moszer, I., Bierne, H. and Cossart, P. (2014). Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity. MBio 5(2): e00969-00914.
  2. Englen, M. D., Valdez, Y. E., Lehnert, N. M. and Lehnert, B. E. (1995). Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells. J Immunol Methods 184(2): 281-283.
  3. Kaplan Zeevi, M., Shafir, N. S., Shaham, S., Friedman, S., Sigal, N., Nir Paz, R., Boneca, I. G. and Herskovits, A. A. (2013). Listeria monocytogenes multidrug resistance transporters and cyclic di-AMP, which contribute to type I interferon induction, play a role in cell wall stress. J Bacteriol 195(23): 5250-5261.
  4. Lobel, L., Sigal, N., Borovok, I., Belitsky, B. R., Sonenshein, A. L. and Herskovits, A. A. (2015). The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA. Mol Microbiol 95(4): 624-644.
  5. Lobel, L., Sigal, N., Borovok, I., Ruppin, E. and Herskovits, A. A. (2012). Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence. PLoS Genet 8(9): e1002887.
  6. Rabinovich, L., Sigal, N., Borovok, I., Nir-Paz, R. and Herskovits, A. A. (2012). Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence. Cell 150(4): 792-802.

简介

在感染期间研究细菌病原体的转录组是一个非常有益的和有效的工具,用于发现有助于成功感染的基因。然而,从感染的细胞或组织中分离细菌RNA是一个挑战性的过程,因为感染细胞裂解物中宿主RNA的量高得多。我们已经优化了用于分离感染骨髓来源的巨噬细胞(BMDM)的单核细胞增生性李斯特菌((单核细胞增生李斯特氏菌)细菌)的RNA的方法。感染后,我们裂解细胞并通过0.45μm过滤器过滤裂解物以丢弃大多数宿主蛋白和RNA。接下来,我们重新悬浮细菌,并在DNase处理后提取RNA。提取的RNA适合于通过实时PCR或微阵列的基因表达分析。我们已经在我们对感染期间的单核细胞增生李斯特氏菌基因调节的体外研究中成功地采用了该方案(Lobel等人,2015; Lobel >,2012; Kaplan Zeevi等人,2013; Rabinovich等人,2012)。

关键字:单核细胞增生李斯特氏菌, 细菌RNA提取, 细胞内的细菌RNA

材料和试剂

  1. 细胞刮刀(Thermo Fisher Scientific,Nunc TM,目录号:179693)
  2. MF-Millipore过滤器(Merck Millipore Corporation,目录号:HAWP04700)
  3. 细胞培养皿(Greiner Bio One International GmbH,目录号:639160)
  4. Eppendorf管(Corning Inc.,Axygen,目录号:MCT175C)
  5. 移液管无菌(25ml)(Greiner Bio One International GmbH)
  6. Falcon管(50ml)(Greiner Bio One International GmbH)
  7. 10403S(Daniel Portnoy的实验室产品)(Becavin ,2014)
  8. 骨髓(来自C57B/6雌性小鼠;订购自Harlan labs Israel)
  9. 液氮
  10. 无RNase的水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  11. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:41965039)
  12. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  13. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM ,目录号:11360088)
  14. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM ,目录号:31350010)
  15. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 庆大霉素(Sigma-Aldrich,目录号:G1397)
  17. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270106)
  18. Dulbecco's磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537)
  19. 脑心浸液(BHI)(默克密理博公司,目录号:1104930500)
  20. 苯酚饱和(pH 4.3)(Thermo Fisher Scientific,目录号:BP1751I-400)
  21. 氯仿(Thermo Fisher Scientific,目录号:BP1145-1)
  22. 异戊醇(Sigma-Aldrich,目录号:W205702)
  23. 无水醋酸钠(Sigma-Aldrich,目录号:W302406)
  24. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
  25. DNase I,无RNA酶(与MnCl 2一起提供)(1U /μl)(Fermentas,目录号:EN0521)
    注意:目前,它是"Thermo Fisher Scientific,目录号:EN0521"。
  26. 10%十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4522)
  27. 乙醇绝对(Merck Millipore Corporation,目录号:1070174000)
  28. M-CSF(L-929条件培养基)(Englen等人,1995)
    注意:或者,M-CSF可购自Sigma(Sigma-Aldrich,目录号:SRP3221)。
  29. BMDM + PS介质(过滤灭菌)(参见配方)
  30. BMDM无PS介质(过滤灭菌)(参见配方)
  31. AE缓冲区(参见配方)
  32. 苯酚 - 氯仿-IAA(参见配方)
  33. 氯仿-IAA(参见配方)

设备

  1. CO 2强制空气培养箱(Thermo Fisher Scientific,型号:3111)
  2. Kontes玻璃杯(Thermo Fisher Scientific,目录号:K953755-0045)
  3. Speed-Vac浓缩器(Thermo Fisher Scientific,目录号:SPD131DDA)
  4. Vortex-Genie 2(Scientific Industries,型号:G560E)
  5. Nanodrop 1000(Thermo Fisher Scientific)
  6. 30℃培养箱(Thermo Fisher Scientific)
  7. 65℃加热块(Thermo Fisher Scientific)
  8. 4℃台式离心机(Thermo Fisher Scientific,Eppendorf TM ,型号:5417R)

程序

  1. 感染
    第1天:
    1. 种子2.0×10 7个骨髓来源的巨噬细胞(BMDM) 用30ml BMDM + PS培养基,每个细菌菌株有3个平板 分析。
    2. 从BHI板中挑取细菌菌落并接种 ?加入到10ml BHI培养基(每个样品重复)中,在30℃温育 孵育,倾斜不摇动过夜。
    第2天:
    1. 在37℃下预温热BMDM无PS培养基和PBS
    2. 用25ml温热的PBS洗涤巨噬细胞单层两次,以除去抗生素。
    3. 加入30 ml新鲜培养基。
    4. 用PBS洗涤1.5ml过夜的细菌培养物两次。
    5. 感染巨噬细胞的MOI为90:1(有利于细菌) 野生型。单核细胞增生李斯特氏菌10403S。当突变体缺陷时 使用细胞内生长,考虑增加MOI
    6. 感染每个板15分钟(与步骤A5相同)。
    7. 在0.5小时孵育后(关于每个样品感染 时间),用30ml PBS洗涤单层两次。加入30 ml预热的BMDM 无PS介质。
    8. 感染后1小时,加入30μl庆大霉素(1:1,000)杀死细胞外细菌
    9. 准备过滤装置和冰无RNase水。
    10. 感染后6小时,收获细菌。单独处理每个板。
      1. 用PBS洗一次单层。
      2. 加入20ml冰冷的无RNA酶的水。
      3. 使用细胞刮刀,通过推动刮刀前面的液体刮擦和溶解细胞
      4. 通过吸取溶解的单层在50ml锥形管中收集。 涡旋30秒。离心2000rpm(720×g/min)/3分钟/4℃
      5. 使上清液通过过滤装置。使用镊子,将滤膜放在15毫升锥形管,松散卷。
      6. 立即通过将15毫升管浸在液氮中冷冻过滤器
      7. 准备下一个样品的过滤器,并重复。

  2. 核酸提取
    第3天:
    1. 制备酸化苯酚:氯仿的1:1混合物,每个样品400μl。 在单独的管中混合,然后排出所得的水层 吸出。
    2. 在冰上解冻含过滤器的管。将每个管完全插入冰中。
    3. 向每个含过滤器的管中加入650μlAE缓冲液。向所有试管中加入缓冲液。
    4. 工作快,大力涡旋你的过滤器包含管这样 过滤器完全搅拌到管的周边和缓冲器 ?在过滤器上洗涤。可能需要额外旋转 同时倒置以完全清洗滤器上的细菌。总是 将管放回冰上,保持管冷。对所有管重复。它可能 可用于在短时间内(1分钟/750rpm/60×g )旋转悬浮液 结束的过程。
    5. 转移含细菌的缓冲液 到含有40μl10%SDS和400μl的1.5ml Eppendorf管中 苯酚/氯仿混合物。对所有管重复。可能需要旋转 ?(1分钟/750转/分钟),得到残留的缓冲液 过滤器
    6. 将所有的管放在多管涡流装置中,全速涡旋10分钟
    7. 孵育管在65℃加热块10分钟。
    8. 以最大速度(14,000RPM/20,817×g )离心5分钟。
    9. 将水层(约400μl)从每个管转移到1.5 ml管,含有40μl3M乙酸钠(pH5.2)和1.0ml 100% 乙醇。彻底涡旋每根管。
    10. 在-80℃1小时孵育样品。
    11. 在4℃以最大速度(14,000RPM/20,817×g )离心20分钟。
    12. 小心地从每个管中吸出乙醇。向每个样品中加入500μl冷的70%乙醇,充分涡旋。
    13. 在4℃以最高速度离心20分钟
    14. 小心地从每个管中吸出乙醇。干燥样品 ?在不加热的速度-Vac装置中约2分钟。它是 关键不要过度干燥你的样品
    15. 向每个样品,加入25微升无RNA酶的水。在室温下孵育20分钟。小心旋转和旋转。
    16. 使用NanoDrop分析样品中的RNA浓度。预期提取约0.5-1μg/板。
    17. 您可以在此阶段联合技术重复。

  3. DNase治疗
    1. 设置反应:
      RNA
      最多2μg(最多44μl)
      10x DNase缓冲区
      5微升
      H sub 2 O
      完成到50微升
      DNase
      1微升
      总计
      50微升
    2. 在37℃孵育45分钟。
    3. 加入450μl无RNase水和500μl苯酚 - 氯仿-IAA混合物, ?通过以最大速度离心2分钟分离各相
    4. 将水层转移到新管中,加入500μl氯仿-IAA 混合,涡旋和分离相,最大2分钟离心 速度
    5. 将水层转移到新管中,加入1ml乙醇和50μl3M乙酸钠(pH5.2)。漩涡
    6. 在-80℃下孵育1小时。
    7. 在4℃以最高速度离心20分钟
    8. 小心地从每个管中吸出乙醇。向每个样品中加入500μl冷的75%乙醇,彻底涡旋。
    9. 在4℃以最高速度离心20分钟
    10. 小心地从每个管中吸出乙醇。在速度Vac 不加热,干燥样品约2分钟。它是 关键不要过度干燥你的样品
    11. 加入12微升无RNA酶的水,在室温下孵育2分钟,涡旋和自旋 - 下降。保持RNA在冰上。
    12. 使用NanoDrop分析样品中的RNA浓度。预期提取约100 ng /样品。

代表数据

我们提供了诱导毒力基因的代表性数据 。单核细胞增生因子在巨噬细胞的细胞内生长期间相比于在BHI培养基中的生长(图1)。在90:1的M.O.I的感染后6小时和在BHI培养基中从细菌生长至中间对数(OD <600 = 0.5),从细胞内细菌纯化mRNA。


图1.在 L生长期间转录 hly 。单核细胞增生李斯特菌在BMDM(6h.p.i.)和BHI实验室培养基中。误差棒代表95%置信区间。结果代表3个重复(N = 3)。 RQ:相对数量

笔记

  1. 检查感染细胞感染前后的形态。细胞应该在感染过程中存活。
  2. 处理膜时要非常小心 - 使用镊子,只触摸边缘。
  3. 当样品通过过滤装置时,首先过滤无RNase的水,以验证真空是否正常,并且系统中没有堵塞。
  4. 你应该观察到DNase I处理后核酸浓度的降低。如果你没有观察到减少,考虑另一轮DNase I治疗

食谱

  1. BMDM + PS培养基(过滤灭菌)
    DMEM
    250 ml
    FBS(在54℃灭活30分钟) 100 ml
    M-CSF(L-929条件培养基)(Englen等人,1995)
    150 ml
    谷氨酰胺
    5 ml
    丙酮酸钠
    5 ml
    β-巯基乙醇 0.5 ml
    Pen/Strep
    5 ml
  2. BMDM无PS介质(过滤灭菌)
    DMEM
    255 ml
    FBS(在54℃灭活30分钟) 100 ml
    M-CSF(L-929条件培养基)(Englen等人,1995)
    150 ml
    谷氨酰胺
    5 ml
    丙酮酸钠
    5 ml
    β-巯基乙醇 0.5 ml
  3. AE缓冲区
    50mM NaOAc(pH 5.2)
    10 mM EDTA
    无RNase水
  4. 4苯酚 - 氯仿-IAA
    苯酚25 ml
    氯仿24ml
    异戊醇1 ml
  5. 氯仿-IAA
    氯仿24ml
    异戊醇1 ml

致谢

该方案改编自以前发表的研究(Lobel等人,2015; Kaplan Zeevi等人,2013; Lobel等人)。 ,2012)。这项工作由以色列科学基金会和ERA-NET PathoGenoMics(由MOH以色列资助)资助AAH。 L.L是由TAU的阿根廷朋友支持。

参考文献

  1. Becavin,C.,Bouchier,C.,Lechat,P.,Archambaud,C.,Creno,S.,Gouin,E.,Wu,Z.,Kuhbacher,A.,Brisse,S.,Pucciarelli,MG,Garcia Dessel,M.,Hain,T.,Portnoy,DA,Chakraborty,T。,Lecuit,M.,Pizarro-Cerda,J.,Moszer,I.,Bierne,H.and Cossart, 。 广泛使用的单核细胞增生性李斯特菌株比较EGD,10403S和EGD- e突显了致病性差异的基因组变异。 MBio 5(2):e00969-00914。
  2. Englen,M.D.,Valdez,Y.E.,Lehnert,N.M.and Lehnert,B.E。(1995)。 粒细胞/巨噬细胞集落刺激因子在鼠L929细胞的培养物中表达和分泌。 J Immunol Methods 184(2):281-283。
  3. Kaplan Zeevi,M.,Shafir,N.S.,Shaham,S.,Friedman,S.,Sigal,N.,Nir Paz,R.,Boneca,I.G.and Herskovits,A.A。(2013)。 单核细胞增生李斯特菌多药耐药转运蛋白和环状二AMP,有助于I型干扰素诱导,在细胞壁应激中起作用。

    195(23):5250-5261。
  4. Lobel,L.,Sigal,N.,Borovok,I.,Belitsky,B.R.,Sonenshein,A.L.和Herskovits,A.A。(2015)。 代谢调节剂CodY通过直接激活单核细胞增生李斯特菌来将单核细胞增生李斯特菌代谢与毒力联系起来毒力调节基因prfA。 Mol Microbiol 95(4):624-644。
  5. Lobel,L.,Sigal,N.,Borovok,I.,Ruppin,E.and Herskovits,A.A。(2012)。 整合基因组分析将异亮氨酸和CodY鉴定为单核细胞增生性李斯特菌的毒力。 PLoS Genet 8(9):e1002887。
  6. Rabinovich,L.,Sigal,N.,Borovok,I.,Nir-Paz,R。和Herskovits,A.A。(2012)。 噬菌体切除激活促进噬菌体逃逸和毒力的<李氏李斯特菌能力基因。 Cell 150(4):792-802。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
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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