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Isolating RNA from the Soil
从土壤中分离RNA   

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Abstract

Next generation sequencing has allowed for the analysis and ability to identify the microbial communities present in the environment. While DNA extraction from environments (such as soil) have provided a wealth of knowledge regarding microbial communities there are drawbacks that one encounters when using DNA as opposed to RNA. RNA allows for the determination of the identity of the microbes that are active and present at a particular time point and thus gives a clear picture of what these microbes are actually doing at a specific point in time and under a set of conditions. Extracting RNA from soil is challenging due to the inherent inhibitors present in the soil such as humic acids. Here we describe modifications to the MoBio RNA PowerSoilTM total RNA isolation kit to reproducively extract total RNA from the soil.

Materials and Reagents

  1. Fresh soil or soil stored at -80 °C
  2. RNA PowerSoilTM Total RNA Isolation Kit (MO BIO Laboratories, catalog number: 12866-25 )
  3. Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (pH 6.7) (Thermo Fisher Scientific, catalog number: BP1752l-400 )
  4. Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758-50mL )
  5. RNase AWAY® (VWR International, catalog number: 72830-022 )
  6. Disposable Gloves
  7. Ice
  8. DEPC water (see Recipes) or nuclease-free water

Equipment

  1. Fisher Vortex Genie 2 (VWR International, model: 12-812 )
  2. Centrifuge (Sorvall® SuperT21 rotor SL-50T) (for 15 ml centrifuge tubes)
  3. Microcentrifuge (VWR International, model: Galaxy 16 )
  4. Shaker (New Brunswick Scientific, model: G-33 )
  5. Incubator set at 45 °C
  6. 5 ml Borosilicated Glass pipette (VWR International, model: 53281-818 )
  7. 1,000 μl pipetman
  8. 200 μl pipetman
  9. 2.5 μl pipetman
  10. -20 °C Freezer
  11. 4 °C Incubator
  12. Microcentrifuge Tube Rack (VWR International, model: CBGTR-080 )
  13. 15 ml falcon tube holder
  14. Laboratory labeling tape (VWR International, catalog number: 89097-902 )
  15. Spectrophotometer (e.g. Thermo Scientific, model: NanoDrop ND-1000 )

Procedure

Note: Use disposable gloves throughout the protocol and change gloves frequently. Spray gloves with RNase Away to remove RNases from your gloves. You can also use a mouth shield to reduce contaminating your sample with RNase.
Protocol is adapted from RNA PowerSoilTM Total RNA Isolation Kit version 02162010.

  1. RNA Isolation
    1. Make DEPC water at least 2 days prior to use.
    2. On the day of your soil RNA extraction make sure to clean your work bench with RNase Away. This includes all pipetman and stands.
    3. Set out the bead tubes from the RNA PowerSoilTM Total RNA Isolation Kit and add the frozen soil that was stored at -80 °C.
    4. Add 2.5 ml of Bead solution to the bead tube and mix solution by vortexing.
    5. Add 0.25 ml of solution labelled SR1 to the bead tube and mix the solution by vortexing.
      Note: Solution at this point should look fairly homogenous and have a muddy consistency.
    6. Add 0.8 ml of solution labelled SR22 to the bead tube and mix the solution by vortexing.
    7. Place tube horizontally on the vortexer and use laboratory label tape to secure the tube on the vortexer.
    8. Vortex tube at maximum speed for 30 min (step modified from original protocol in order to more readily and efficiently remove non-DNA material that would interfere with RNA isolation and lyse cells).
    9. Remove laboratory label tape from the tube and vortexer and place tube in the stand.
    10. Using the 5 ml borosilicated glass pipet add 3.5 ml of phenol: chloroform: isoamyl alcohol, pH 6.7 and mix solution by inverting up and down 10 times.
    11. Secure tube horizontally on a shaker and shake solution for 30 min at 200 rpm (step modified from original protocol in order to prevent RNA shearing that would affect RNA integrity and concentration).
    12. Remove bead tube from the shaker and centrifuge at room temperature for 10 min at 2,500 x g.
    13. Transfer the aqueous (upper) layer using a 1,000 μl pipetman to a new clean 15 ml falcon tube provided in the RNA PowerSoilTM Total RNA Isolation Kit. Ensure that you only take the top layer and it may be best to leave behind some of the aqueous layer to ensure no interphase or lower layer is transferred.
    14. Add 1.5 ml of solution labelled SR3 to the new tube containing the upper aqueous phase. Close tube and invert 10 times.
    15. Incubate the tube for 10 min at 4 °C.
    16. Centrifuge tube at room temperature for 10 min at 2,500 x g.
    17. Sometimes a pellet is formed after this step thus just in case transfer the supernatant to a new 15 ml falcon tube provided using a 1,000 μl pipetman.
    18. Using a 5 ml borosilicated glass pipet transfer 5 ml of solution SR4 to the new collection tube. Close tube and invert 10 times.
    19. Incubate tube at -20 °C for 30 min.
    20. Centrifuge tube at room temperature for 30 min at 2,500 x g.
    21. Decant the supernatant.
    22. Re-centrifuge the tubes at room temperature for 5 min at 2,500 x g.
    23. Using a 1,000 μl pipetman remove any remaining supernatant trying not to disturb the pellet.
    24. Allow tubes to dry for 5 min.
      Note: Pellet may be white, gray or light brown in color.

  2. RNA purification
    1. Shake solution SR5.
    2. Once pellet has dried add 1 ml of solution SR5 to the tube and mix by inverting and shaking solution.
    3. If pellet has not dissolved in solution SR5 incubate at 45 °C and check on the tube every 2 min and shake solution.
    4. Repeat until pellet has dissolved in solution SR5.
      Note: Solution may be clear or tan in color.
    5. After pellet has dissolved place one RNA capture column in a new 15 ml falcon tube provided.
    6. Shake solution SR5 and add 2 ml of solution SR5 to the RNA capture column.
    7. Allow solution SR5 to gravity flow through the column.
    8. Once solution SR5 has reached the top of the resin in the column add the dissolved pellet to the column and allow it to gravity flow through the column.
    9. Once the dissolved pellet has reached the top of the column resin shake solution SR5 and add 1 ml of solution SR5 to the column and allow it to gravity flow through the column.
    10. Once solution SR5 has reached the top of the column resin, shake solution SR6 and add 1 ml to the column.
    11. Immediately transfer the column to a new 15 ml falcon tube.
    12. Allow solution SR6 to flow through the column.
    13. Transfer the eluted RNA to a 2.2 ml collection tube provided in the kit.
    14. Add 1 ml of solution SR4 to the 2.2 ml collection tube and invert tube to mix 10 times.
    15. Incubate tube at -20 °C for 20 min.
    16. Centrifuge the tube at 4 °C for 15 min at 13,000 x g.
    17. You should be able to see a pellet on the side of your tube. The pellet may be white or tan in color.
    18. Decant the supernatant.
    19. Centrifuge the tube at 4 °C for 5 min at 13,000 x g.
    20. Using a 200 μl pipetman carefully without touching the pellet remove the remaining supernatant from the tube.
    21. Allow the tube to dry in air for 5 min.

  3. Evaluation of RNA samples
    1. Re-suspend the RNA pellet in 30 μl of solution SR7.
    2. Place collection tube on ice.
    3. Determine concentration of RNA using a spectrophotometer for RNA concentration can also monitor A260/280 to determine RNA integrity (if A260/280 between 1.8-2.0 RNA is of good quality for further downstream applications).
    4. Check RNA integrity via the use of an agarose gel.

Recipes

  1. DEPC Water
    Add 1 ml of diethyl pyrocarbonate per liter of Millipore water
    Stir solution overnight
    Autoclave solution for 30 min in liquid cycle
    Let solution cool to room temperature
    Store at room temperature 

Acknowledgments

Work was supported by a grant from the National Science Foundation to J.M.V. (MCB-0950857).

References

  1. Chaparro, J. M., Badri, D. V., Bakker, M. G., Sugiyama, A., Manter, D. K. and Vivanco, J. M. (2013). Root exudation of phytochemicals in Arabidopsis follows specific patterns that are developmentally programmed and correlate with soil microbial functions. PLoS One 8(2): e55731.
  2. MoBio RNA PowerSoil® Total RNA Isolation Kit instruction manual version 02162010

简介

下一代测序允许分析和鉴定存在于环境中的微生物群落的能力。 虽然从环境(例如土壤)中提取DNA提供了关于微生物群落的丰富知识,但是当使用DNA而不是RNA时会遇到缺点。 RNA允许测定活性并存在于特定时间点的微生物的身份,并因此给出这些微生物实际上在特定时间点和在一组条件下进行的清楚图片。 从土壤中提取RNA是具有挑战性的,因为存在于土壤中的固有抑制剂如腐殖酸。 在这里我们描述修改的MoBio RNA PowerSoil TM 总RNA分离试剂盒从土壤中重复提取总RNA。

材料和试剂

  1. 新鲜土壤或土壤储存在-80°C
  2. RNA PowerSoil Total RNA Isolation Kit(MO BIO Laboratories,目录号:12866-25)
  3. 苯酚/氯仿/异戊醇25:24:1(pH6.7)(Thermo Fisher Scientific,目录号:BP17521-400)
  4. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758-50mL)
  5. RNase AWAY ®(VWR International,目录号:72830-022)
  6. 一次性手套
  7. 冰块
  8. DEPC水(见Recipes)或无核酸酶水

设备

  1. Fisher Vortex Genie 2(VWR International,型号:12-812)
  2. 离心机(Sorvall SuperT21转子SL-50T)(用于15ml离心管)
  3. 微量离心机(VWR International,型号:Galaxy 16)
  4. Shaker(New Brunswick Scientific,型号:G-33)
  5. 孵育器设置在45°C
  6. 5ml硼硅玻璃移液管(VWR International,型号:53281-818)
  7. 1000μl移液器
  8. 200μl移液器
  9. 2.5μl移液器
  10. -20°C冰箱
  11. 4°C孵育器
  12. 微量离心机管架(VWR International,型号:CBGTR-080)
  13. 15 ml falcon管夹
  14. 实验室标签带(VWR International,目录号:89097-902)
  15. 分光光度计(例如Thermo Scientific,型号:NanoDrop ND-1000)

程序

注意:在整个方案中使用一次性手套,并经常更换手套。 带RNase的喷手戴手套去除RNases。 您还可以使用口罩来减少RNase对样品的污染。

总RNA分离试剂盒版本02162010。


  1. RNA分离
    1. 使DEPC水至少2天前使用。
    2. 在你的土壤RNA提取的一天,确保用RNase Away清洁你的工作台。 这包括所有的移液器和支架
    3. 设置从RNA PowerSoil TM 总RNA分离试剂盒的珠管,并添加存储在-80℃的冻土。
    4. 向珠子管中加入2.5ml珠子溶液,并通过涡旋混合溶液。
    5. 向珠子管中加入0.25ml标记为SR1的溶液,并通过涡旋混合溶液 注意:此时的解决方案应该看起来很均匀,并且具有泥泞的一致性。
    6. 向珠管中加入0.8ml标记为SR22的溶液,并通过涡旋混合溶液
    7. 将管子水平放置在涡流器上,并使用实验室标签带将管子固定在涡流器上
    8. 涡旋管以最大速度30分钟(从原始方案修改步骤,以便更容易和有效地去除会干扰RNA分离和裂解细胞的非DNA材料)。
    9. 从试管和涡流器中取出实验室标签带,将试管放在支架上
    10. 使用5ml硼硅玻璃吸管,加入3.5ml苯酚:氯仿:异戊醇,pH6.7,通过上下颠倒10次混合溶液。
    11. 将管子在振荡器上水平固定,并以200rpm摇动溶液30分钟(从原始方案修改的步骤,以防止RNA剪切,影响RNA完整性和浓度)。
    12. 从摇床上取下胎管,并在室温下以2,500×g离心10分钟。
    13. 使用1000微升移液器转移水(上)层到新的干净的15毫升falcon管提供在RNA PowerSoil总RNA分离试剂盒。确保你只采取顶层 并且最好留下一些水层以确保没有相间或下层转移。
    14. 向含有上层水相的新管中加入1.5ml标记为SR3的溶液。 关闭管并翻转10次
    15. 在4℃下孵育10分钟。
    16. 离心管在室温下以2,500×g离心10分钟。
    17. 有时在此步骤后形成沉淀,因此只是为了将上清液转移到使用1000μl移液管提供的新的15ml falcon管中。
    18. 使用5毫升硼硅玻璃移液管转移5毫升溶液SR4到新的收集管。 关闭管并翻转10次
    19. 将管在-20℃下孵育30分钟
    20. 离心管在室温下以2,500×g离心30分钟。
    21. 倾析上清液。
    22. 在室温下以2,500×g再次离心该管5分钟。
    23. 使用1000μl移液管除去任何残留的上清液,不要打扰沉淀
    24. 让管子干燥5分钟。
      注意:颗粒可能是白色,灰色或浅棕色。

  2. RNA纯化
    1. 摇动溶液SR5。
    2. 一旦颗粒干燥,向管中加入1ml溶液SR5,通过颠倒和摇动溶液混合
    3. 如果沉淀物未溶解于溶液中,SR5在45℃下孵育并每2分钟检查管子并摇动溶液。
    4. 重复,直到颗粒溶解在SR5溶液中 注意:溶液可以是透明的或棕褐色的。
    5. 颗粒溶解后,在一个新的15ml falcon管中提供一个RNA捕获柱
    6. 摇动溶液SR5,并将2ml溶液SR5加入RNA捕获柱
    7. 使溶液SR5重力流过色谱柱。
    8. 一旦溶液SR5已经到达柱中树脂的顶部,将溶解的沉淀物加入柱中并允许其重力流过柱。
    9. 一旦溶解的颗粒已经达到柱树脂摇动溶液SR5的顶部,并向柱中加入1ml溶液SR5,并允许其重力流过柱。
    10. 一旦溶液SR5已达到柱树脂的顶部,摇动溶液SR6,并向柱中加入1ml。
    11. 立即将色谱柱转移到新的15 ml falcon管中。
    12. 使溶液SR6流过该柱。
    13. 将洗脱的RNA转移到试剂盒提供的2.2ml收集管中
    14. 加入1毫升溶液SR4到2.2毫升收集管和倒转管混合10次
    15. 将管在-20℃下孵育20分钟
    16. 将管在4℃下以13,000×g离心15分钟。
    17. 你应该能够在管子的一边看到一颗颗粒。 颗粒可以是白色或棕褐色。
    18. 倾析上清液。
    19. 在4℃下以13,000×g离心管5分钟。
    20. 小心地使用200μl移液管而不接触沉淀物,从管中除去剩余的上清液
    21. 让管在空气中干燥5分钟。

  3. RNA样品的评价
    1. 将RNA沉淀重悬在30μl溶液SR7中。
    2. 将收集管放在冰上。
    3. 使用分光光度计确定RNA的浓度,用于RNA浓度也可以监测A 260/280以确定RNA完整性(如果1.8-2.0 RNA之间的A 260/280是高质量的 进一步下游应用)。
    4. 通过使用琼脂糖凝胶检查RNA完整性。

食谱

  1. DEPC水
    每升Millipore水中加入1ml焦碳酸二乙酯
    搅拌溶液过夜
    在液体循环中高压灭菌溶液30分钟
    让溶液冷却至室温
    储存在室温下 

致谢

工作得到国家科学基金会授予J.M.V.的资助。 (MCB-0950857)。

参考文献

  1. Chaparro,J.M.,Badri,D.V.,Bakker,M.G.,Sugiyama,A.,Manter,D.K。和Vivanco,J.M。(2013)。 拟南芥中植物化学物质的根渗出 遵循具有发育程序并与土壤微生物功能相关的特定模式。

    8(2):e55731。
  2. MoBio RNA PowerSoil ® Total RNA Isolation Kit使用手册 版本02162010
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chaparro, J. M. and Vivanco, J. M. (2013). Isolating RNA from the Soil. Bio-protocol 3(18): e903. DOI: 10.21769/BioProtoc.903.
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