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Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation
通过氯化锂沉淀法纯化DSS处理的鼠组织的总RNA   

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参见作者原研究论文

本实验方案简略版
BMC Research Notes
Sep 2013

Abstract

We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.

Keywords: Dextran sodium sulfate (葡聚糖硫酸钠), Colitis (结肠炎), Lithium chloride (氯化锂), RNA (RNA), q-RT-PCR (q-RT-PCR)

Background

Dextran Sulfate Sodium (DSS) is very commonly used in laboratories to induce colitis in rodents. Specifically, it mimics the clinical and histological features of human Inflammatory Bowel Disease (IBD) with Ulcerative Colitis (UC) characteristics. DSS is diluted in the drinking water and penetrates tissues. We have observed that contamination of RNA extracts with DSS prevented successful subsequent amplification processes from the colon and small intestine, but also blood and other tissues obtained from DSS-treated animals. We had previously shown that the presence of DSS in the samples inhibited reverse transcription and polymerase chain reaction amplification (Viennois et al., 2013). This inhibitory effect was observed in a dose depended manner by Kerr et al. and they suggested a poly-A-purification based technique to remove DSS from total RNA extract (Kerr et al., 2012). We hereby propose another efficient and economical method for purifying total RNA extracts from DSS traces based on lithium chloride (LiCl) precipitations. This method has been extensively used in our laboratory as well as in others (Chassaing et al., 2012, Li et al., 2016); however, no attempt has been taken to document the procedure in detail. Therefore, we provide a detailed description of LiCl purification procedure of total RNA primarily isolated from DSS-treated murine tissue with another method (Trizol, Spin column-based nucleic acid purification…).

Materials and Reagents

  1. Pipette tips (0.1-10 µl, 1-200 µl, 100-1,000 µl)
  2. Eppendorf Safe-Lock Tubes, 1.5 ml (Eppendorf, catalog number: 022363204 )
  3. 8 M lithium chloride (LiCl) (SIGMA Lithium Chloride Solution, 8 M Solution, Sigma-Aldrich, catalog number: L7026-100ML , 090M8728)
  4. RT-PCR Grade Water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9935 )
  5. Pure 100% ethanol (Decon Labs, catalog number: 2716 )
  6. 3 M sodium acetate, pH 5.2 (see Recipes)
  7. 70% ethanol (see Recipes)

Equipment

  1. Pipettes 0.5-10 µl, 10-100 µl and 100-1,000 µl (Eppendorf, model: Research® plus , Variable Adjustable Volume Pipettes)
  2. Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM Micro 21R , or equivalent)
  3. Multi-Mode Microplate Reader (BioTek Instruments, model: BioTekTM SynergyTM 2 )
  4. -20 °C freezer

Procedure

  1. RNAs are in solution in RNase-free water after previous isolation with any method (Trizol, Spin column-based nucleic acid purification).
  2. Add 0.1 volume of 8 M LiCl solution to 1 volume of RNA solution (i.e., 5 μl of 8 M LiCl for 50 μl RNA solution).
  3. Mix well by pipetting up and down and incubate on ice for 2 h.
  4. After the incubation, centrifuge at 14,000 x g for 30 min, at 4 °C.
  5. Discard the supernatant and dissolve the pellet (that might be almost invisible depending on the initial quantity of RNA) in 200 μl RNase-free water.
  6. Repeat steps from 1 to 5. Briefly, perform a second precipitation with 0.1 volume of LiCl (20 μl), mix by pipetting and incubate the solution for 2 h on ice, centrifuge and dissolve the pellet in 200 μl of water.
  7. Add 0.1 volume (i.e., 20 μl) of 3 M sodium acetate (pH = 5.2) and 2.0 volumes (i.e., 400 μl) of -20 °C prechilled 100% ethanol to 1 volume of RNA solution. Incubate at -20 °C for 30 min.
  8. Centrifuge at 14,000 x g for 30 min at 4 °C.
  9. Discard the supernatant and wash the pellet with 100 μl of -20 °C prechilled 70% ethanol.
  10. Centrifuge at 14,000 x g for 10 min at 4 °C.
  11. Remove the supernatant carefully with a pipet without disturbing the pellet which might be invisible at this step. 
    Note: The supernatant should be removed carefully. The expected location of the pellet (which is determined by the position of the tube in the centrifuge) must be considered for pipetting up the supernatant.
  12. Let the pellet air dry at room temperature for 5-10 min.
  13. Dissolve the pellet in RNase free water in a volume smaller or equal to the initial volume of RNA solution in Step 1.
  14. Determine RNA yield by measuring the absorbance peak at 260 nm with a multi-mode microplate reader. The absorbance ratio at 260/280 nm is also determined to evaluate the purity of RNA with an acceptable ratio from 2 to 2.2.
  15. RNAs from DSS-treated tissues can now be used for cDNA synthesis and qPCR according to standard protocols.

Data analysis

Agarose gel electrophoresis demonstrates that, before LiCl precipitation, housekeeping gene 36B4 cDNA amplification products are obtained from non-DSS-treated mice as indicated by a clear band while amplification is inhibited in DSS-treated mice as indicated by the smear (Figure 1). After LiCl precipitation, cDNA obtained from non-DSS and DSS treated samples were both successfully amplified, as attested by a distinct band (Figure 1).


Figure 1. LiCl purification allows amplification of cDNA from DSS-treated tissues. Total RNAs were extracted from colonic tissues obtained from mice treated with DSS diluted at 3% in the drinking water or water controls. After cDNA synthesis, qPCR for the housekeeping gene 36B4 was performed and the product of amplification was visualized by electrophoresis. The presence of a smear indicates that the amplification of 36B4 cDNA was inhibited in DSS-treated mice before LiCl purification. After purification of the DSS- and non-DSS RNA samples with LiCl, clear bands indicate that 36B4 cDNA was successfully amplified from both DSS- and non-DSS samples.

Notes

  1. This protocol might be associated with a decrease of RNA yield. The volume of re-suspension in Step 13 might be adjusted according to the initial volume and should be the same or smaller (if the RNA should be at a similar or higher concentration than before the LiCl purification).
  2. Due to the partial loss of RNA that might occur during the successive precipitations, the protocol should be performed on RNA samples with an initial concentration of no less than 200-300 ng/μl. The best results are obtained on samples from 500 ng/μl initial RNA concentration and above.
  3. Within the same experiment, all groups of samples, whether they were obtained from mice administered with DSS or from the water control group, should be equally subjected to the LiCl purification process.

Recipes

  1. 3 M sodium acetate, pH 5.2 (for 100 ml)
    24.61 g of sodium acetate
    100 ml of Milli-Q® ultra-pure water
    Adjust pH to 5.2 with HCl
  2. 70% ethanol
    70% of 100% ethanol
    30% of Milli-Q® ultra-pure water

Acknowledgments

EV is a recipient of the Career Development Award from the Crohn’s and Colitis Foundation. DM is a recipient of a Research Scientist Award from the Department of Veteran Affairs. This work was supported by grants from the National Institutes of Health of Diabetes and Digestive and Kidney (DK116306, DK107739; DK071594 to DM). We thank Samantha Spencer for proofreading the manuscript. This protocol was adapted from published work by Cathala et al. (1983) and Viennois et al. (2013). The authors declare no conflicts of interest within this work.

References

  1. Cathala, G., Savouret, J. F., Mendez, B., West, B. L., Karin, M., Martial, J. A. and Baxter, J. D. (1983). A method for isolation of intact, translationally active ribonucleic acid. DNA. 2(4):329-35.
  2. Chassaing, B., Srinivasan, G., Delgado, M. A., Young, A. N., Gewirtz, A. T. and Vijay-Kumar, M. (2012). Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation. PLoS One 7(9): e44328.
  3. Kerr, T. A., Ciorba, M. A., Matsumoto, H., Davis, V. R., Luo, J., Kennedy, S., Xie, Y., Shaker, A., Dieckgraefe, B. K. and Davidson, N. O. (2012). Dextran sodium sulfate inhibition of real-time polymerase chain reaction amplification: a poly-A purification solution. Inflamm Bowel Dis 18(2): 344-348.
  4. Li, Y. H., Xiao, H. T., Hu, D. D., Fatima, S., Lin, C. Y., Mu, H. X., Lee, N. P. and Bian, Z. X. (2016). Berberine ameliorates chronic relapsing dextran sulfate sodium-induced colitis in C57BL/6 mice by suppressing Th17 responses. Pharmacol Res 110: 227-239.
  5. Viennois, E., Chen, F., Laroui, H., Baker, M. T. and Merlin, D. (2013). Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA. BMC Res Notes 6: 360.

简介

我们开发了一种从DSS(葡聚糖硫酸钠)处理的小鼠组织中纯化RNA的方案。 这种防止DSS处理组织中观察到的q-RT-PCR下游抑制的方法依赖于氯化锂的连续沉淀。

【背景】葡聚糖硫酸钠(DSS)在实验室中非常普遍地用于诱导啮齿动物的结肠炎。具体而言,它模拟人类炎性肠病(IBD)与溃疡性结肠炎(UC)特征的临床和组织学特征。 DSS在饮用水中稀释并穿透组织。我们已经观察到用DSS污染RNA提取物阻止了从结肠和小肠成功的后续扩增过程,但也阻止了从DSS处理的动物获得的血液和其他组织。之前我们已经证明,样品中DSS的存在抑制了逆转录和聚合酶链式反应扩增(Viennois等人,2013)。 Kerr等人以剂量依赖的方式观察到这种抑制作用,他们提出了基于多聚-A纯化的技术以从总RNA提取物中去除DSS(Kerr等人,,2012)。我们在此提出了另一种有效和经济的方法,用于基于氯化锂(LiCl)沉淀纯化来自DSS迹线的总RNA提取物。这种方法已经在我们的实验室以及其他方面得到了广泛的应用(Chassaing et al。,2012,Li et al。,2016)。但是,没有尝试详细记录该程序。因此,我们详细描述了用另一种方法(Trizol,Spin柱为基础的核酸纯化)从主要从DSS处理的鼠组织中分离的总RNA的LiCl纯化步骤。

关键字:葡聚糖硫酸钠, 结肠炎, 氯化锂, RNA, q-RT-PCR

材料和试剂

  1. 移液器吸头(0.1-10μl,1-200μl,100-1,000μl)
  2. Eppendorf Safe-Lock Tubes,1.5毫升(Eppendorf,产品目录号:022363204)
  3. 8M氯化锂(LiCl)(SIGMA氯化锂溶液,8M溶液,Sigma-Aldrich,目录号:L7026-100ML,090M8728)
  4. RT-PCR等级水(Thermo Fisher Scientific,Ambion TM,目录号:AM9935)
  5. 纯100%乙醇(Decon Labs,目录号:2716)
  6. 3M醋酸钠,pH 5.2(见食谱)
  7. 70%乙醇(见食谱)

设备

  1. 移液器0.5-10μl,10-100μl和100-1,000μl(Eppendorf,型号:Research plus plus,可变可调容量移液器)
  2. 冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM Legend TM Micro 21R,或同等产品)
  3. 多模式酶标仪(BioTek Instruments,型号:BioTek TM Synergy TM 2)
  4. -20°C冷冻机

程序

  1. 先前用任何方法分离(Trizol,基于旋转柱的核酸纯化)后,RNA在无RNA酶的水中溶解。
  2. 将0.1体积的8M LiCl溶液加入到1体积的RNA溶液中(即,5μl的8M LiCl用于50μlRNA溶液)。

  3. 上下搅拌,并在冰上孵育2小时
  4. 温育后,在4℃下以14,000×gg离心30分钟。
  5. 丢弃上清液并在200μl无RNase的水中溶解沉淀(根据RNA的初始数量可能几乎看不见)。
  6. 重复步骤从1到5.简言之,用0.1体积的LiCl(20μl)进行第二次沉淀,通过移液混合并将溶液在冰上孵育2h,离心并将沉淀溶解在200μl水中。
  7. 加入预冷的100%-20℃预冷的3体积乙酸钠(pH = 5.2)和2.0体积(em,400μl)的0.1体积(即20μl)乙醇至1体积的RNA溶液。在-20°C孵育30分钟。

  8. 在14,000 xg g离心30分钟,4℃。
  9. 弃去上清液,用100μl-20℃预冷的70%乙醇洗涤沉淀。

  10. 14,000×g g离心10分钟

  11. 使用移液器小心移除上清液,而不会干扰在此步骤中可能看不到的沉淀。 
    注意:应仔细清除上清液。必须考虑颗粒的预期位置(由离心机中管的位置决定),以吸取上清液。

  12. 在室温下让颗粒风干5-10分钟。

  13. 在小于或等于步骤1中RNA溶液初始体积的体积中将沉淀溶解于无RNase的水中。
  14. 用多模式酶标仪测量260 nm处的吸光度峰值,确定RNA产量。还测定260/280nm处的吸光度比率以评估RNA纯度,其中可接受比例为2至2.2。
  15. 根据标准方案,来自DSS处理的组织的RNA现在可以用于cDNA合成和qPCR。

数据分析

琼脂糖凝胶电泳表明,在LiCl沉淀之前,如由清除条带所示,从非DSS处理的小鼠获得管家基因36B4 cDNA扩增产物,而如涂片所示,DSS处理的小鼠中的扩增受到抑制(图1)。 LiCl沉淀后,从非DSS和DSS处理的样品获得的cDNA都被成功扩增,如通过不同的条带所证明的(图1)。


图1.LiCl纯化允许从DSS处理的组织中扩增cDNA。从饮用水或水对照中稀释为3%的DSS处理的小鼠获得的结肠组织中提取总RNA。 cDNA合成后,进行持家基因36B4的qPCR,并通过电泳显现扩增产物。涂片的存在表明在LiCl纯化前DSS处理的小鼠中36B4 cDNA的扩增受到抑制。用LiCl纯化DSS和非DSS RNA样品后,清晰的条带表明36B4 cDNA成功地从DSS和非DSS样品扩增。

笔记

  1. 该协议可能与RNA产量的降低有关。步骤13中的再悬浮体积可根据初始体积进行调整,并且应该相同或更小(如果RNA应该处于与LiCl纯化前相似或更高的浓度)。
  2. 由于在连续沉淀过程中可能发生RNA的部分丢失,应该对初始浓度不低于200-300 ng /μl的RNA样品进行操作。从500 ng /μl初始RNA浓度及以上的样品中获得最佳结果。
  3. 在同一实验中,所有样品组,不管它们是从给予DSS的小鼠还是从水对照组获得的,都应同样进行LiCl纯化过程。

食谱

  1. 3M醋酸钠,pH 5.2(100毫升)

    24.61克醋酸钠 100毫升Milli-Q®超纯水
    用HCl将pH值调节至5.2
  2. 70%乙醇
    70%的100%乙醇
    Milli-Q®超纯水的30%

致谢

E.V是克罗恩和大肠炎基金会职业发展奖的获得者。我们感谢萨曼莎斯宾塞校对手稿。该协议改编自Cathala等人(1983)和Viennois等人(2013)的已发表的工作。作者声明在这项工作中没有利益冲突。

参考

  1. Cathala,G.,Savouret,J.F.,Mendez,B.,West,B.L.,Karin,M.,Martial,J.A。和Baxter,J.D。(1983)。 分离完整,翻译活性核糖核酸的方法 DNA 。 2(4):329-35。
  2. Chassaing,B.,Srinivasan,G.,Delgado,M.A.,Young,A.N。,Gewirtz,A.T。和Vijay-Kumar,M.(2012)。 粪便脂质运载蛋白2,一种敏感且广泛动态的肠道炎症非侵入性生物标志物。 PLoS One 7(9):e44328。
  3. Kerr,T. A.,Ciorba,M.A.,Matsumoto,H.,Davis,V.R.,Luo,J.,Kennedy,S.,Xie,Y。,Shaker,A.,Dieckgraefe,B.K。和Davidson,N.O。(2012)。 葡聚糖硫酸钠抑制实时聚合酶链式反应扩增:一种poly-A纯化溶液。< / a> Inflamm Bowel Dis 18(2):344-348。
  4. Li,Y.H.,Xiao,H.T.,Hu,D.D.,Fatima,S.,Lin,C.Y.,Mu,H.X.,Lee,N.P.and Bian,Z.X.(2016)。 小檗碱通过抑制Th17应答来改善C57BL / 6小鼠慢性复发性葡聚糖硫酸钠诱导的结肠炎。 / a> Pharmacol Res 110:227-239。
  5. Viennois,E.,Chen,F.,Laroui,H.,Baker,M.T。和Merlin,D。(2013)。 葡聚糖硫酸钠抑制聚合酶和逆转录酶的活性:氯化锂纯化,快速高效技术来纯化RNA。 BMC Res Notes 6:360.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Viennois, E., Tahsin, A. and Merlin, D. (2018). Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation. Bio-protocol 8(9): e2829. DOI: 10.21769/BioProtoc.2829.
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