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RNA Isolation From Meloidogyne Spp. Galls
根结线虫(Meloidogyne Spp)瘤中RNA的分离   

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Abstract

We describe an efficient method to obtain a sufficient quantity of RNA from nematode-induced galls with a high quality and integrity, proved to be appropriate for transcriptomic analysis, i.e. real time PCR, microarray hybridization or second generation sequencing. This protocol is efficient for small quantities of galls (organs with high protein and sugar contents). The protocol allows obtaining an RNA yield of 5-15 μg total RNA from 250-300 hand dissected galls at 3 days post infection (dpi) (Figure 1). It was proved particularly for Arabidopsis and tomato.

Keywords: Galls(瘿), Meloidogyne(根结线虫), Efficient RNA extraction(高效RNA提取), Transcriptomics(transcriptomics), High quality RNa(高质量RNA)


Figure 1. 3 dpi Arabidopsis gall from M. javanica. Blue lines indicate the collected material, galls with a small portion of roots (that allows easy handling), frozen and processed for RNA extraction.

Materials and Reagents

  1. Arabidopsis infected by M. javanica (3 dpi or 7 dpi)
  2. TRI Reagent (Molecular Research Centre, MRC, catalog number: TR-118 )
  3. Chloroform (Scharlau, catalog number: CL02031000 )
  4. Isopropanol (Merck KGaA, catalog number: 0 9634 )
  5. Sodium citrate (Sigma-Aldrich, catalog number: s4641 )
  6. Sodium chloride (Duchefa, catalog number: S0520 )
  7. NaOH (Duchefa, catalog number: s0523 )
  8. Ethanol (Merck KGaA, catalog number: 100986 )
  9. Diethylpyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 )
  10. RNase-free water
  11. Acetone (Thermo Fisher Scientific, catalog number: A/0600/17 )
  12. Liquid nitrogen
  13. RNeasy Mini Kit (QIAGEN, catalog number: 74104 ))
  14. RNase-Free DNase Set (QIAGEN, catalog number: 79254 )
  15. High salt precipitation solution (see Recipes)

Equipment

  1. Centrifuges
  2. Microfuge
  3. 1.5 ml Eppendorf® tubes
  4. Porcelain mortar and pestles, textured surface on bowl interior, 60 mm diameter
  5. Fume hood

Procedure

     Note: Use gloves and goggles for all procedure.

  1. Gall collection: To perform the RNA isolation from galls with a good efficiency, 250-300 hand dissected galls at 3 days post infection (3 dpi) or 100-150 galls at 7 dpi, should be collected. Collect galls and rapidly freeze them to a 1.5 ml Eppendorf® tube in liquid nitrogen.
    Note: If scaled down, the RNA yield is not proportional to the amount of tissue. You can accumulate galls from independent collection events in the same Eppendorf®.

  2. Store at -80 °C until further processing (step C).

  3. Homogenization:
    1. Clean mortar and pestles with acetone, let them dry on the bench and autoclaved to avoid RNase activity. CAUTION: Check that mortar, pestle and pipettes used during the extraction have not been in contact with RNases. Sometimes it is preferable to keep material exclusively for RNA extraction.
    2. Add liquid nitrogen into the mortar with the pestle to cold them down.
    3. Add the galls into the mortar and proceed into a fume hood.
    4. When liquid nitrogen is nearly evaporated, add TRI Reagent® (500-750 μl/200-300 galls at 3 dpi); the total volume should not exceed 10% of the volume of TRI Reagent used for homogenization, as suggested by the TRI Reagent® protocol.
    5. Grind galls with a mortar and pestle. TRI Reagent solution initially becomes frozen, but it will melt gradually as a result of the homogenization. Proceed until a liquid appearance is observed and no visible tissue pieces are identified within the Reagent solution.
      Note: Do not leave traces of galls without homogenizing, since this step can be limiting for good efficiency.
    6. Transfer the gall homogenate with a pipette to a 1.5 ml Eppendorf® tube and place it on ice while you homogenize the rest of the samples.

  4. Phase separation
    1. Centrifuge at 12,000 x g for 10 min at 4 °C and transfer the supernatant containing the RNA to a new 1.5 ml Eppendorf® tube.
      Note: The resulting pellet contains membranes, polysaccharides and high molecular weight DNA while the supernatant contains RNA. All insoluble material should be removed from the homogenate.
    2. Add 100 μl of chloroform per 500 μl of TRl Reagent® in a fume hood. Cap sample tubes securely.
    3. Shake tubes vigorously by hand for 15 s and incubate them at room temperature (RT) for 5 to 10 min.
      Note: Do not use vortex.
    4. Centrifuge the samples at 12,000 x g for 15 min at 4 °C.
    5. Transfer the upper aqueous phase to a new tube.

  5. RNA precipitation
    1. Add 125 μl of isopropanol and 125 μl of high salt buffer per 500 μl of TRl Reagent®. Mix the solution and store it for 5-10 min at RT.
      Note: The high salt precipitation solution removes contaminating compounds as proteoglycan and polysaccharide from the isolated RNA.
    2. Centrifuge at 12,000 x g for 8 min at 4 °C.
    3. Discard the supernatant.

  6. RNA Wash
    1. Wash the RNA pellet with 1 ml of 75% ethanol and mix by inversion several times.
    2. Centrifuge at 7,500 x g for 5 min at 4 °C and discard the supernatant.
    3. Repeat steps E-1 and 2.
    4. Spin 10 sec in a microfuge and discard carefully the remaining liquid.
    5. Air dry the pellet for 3-5 min. Do not let the RNA over-dry, as this will make it difficult to dissolve. The white RNA pellet will turn clear when it dries out.
    6. Add 100 μl DEPC-water or RNase-free water immediately after the pellet becomes clear.
    7. Dissolve RNA by incubating for 10 min at 37 °C followed by 5 min at 60 °C.
    8. Store RNA solution at -80 °C or continue with further RNA Cleanup by DNase digestion.

  7. Combined in column DNase I Digestion and RNA Cleanup. This procedure is based on the combined use of two different commercial kits (RNase-Free DNase Set and RNeasy Mini Kit). Please, use handling instructions of all buffers and reagents following their recommendations.
    Note: Buffers as RPE, RTL, RW1, RDD are supplied by the companies within the kits. Before starting RNA cleanup, make sure the DNase I and Buffer RPE are ready to use.
    1. Preparation of DNase I stock solution: Inject 550 μl RNase-free water into the lyophilized DNase I vial using an RNase-free needle and syringe. Mix gently by inverting the vial and divide it in 10 μl single-use aliquots. Store at -20 °C for up 9 months.
    2. Preparation of Buffer RPE: Add 4 volumes of 96-100% ethanol (44 ml) as indicated in the bottle.
    3. Adjust the RNA sample volume to 100 μl with RNase-free water.
    4. Add 350 μl of Buffer RLT and mix well by pipetting.
    5. Add 250 μl EtOH, mix well by pipetting.
    6. Transfer the sample to an RNeasy Mini spin column placed in a 2 ml collection tube. Close the lid gently and centrifuge at 8,000 x g for 15 sec. Discard the flow-through.
    7. Add 350 μl buffer RW1 to the RNeasy spin column. Close the lid gently and centrifuge at 8,000 x g for 15 sec. Discard the flow-through.
    8. Add 70 μl buffer RDD (supplied with the RNase-Free DNase Set) to 10 μl DNase I stock solution. Mix gently by inverting the tube.
      Note: Do not vortex.
    9. Add 80 μl of the DNase I incubation Mix to the RNeasy spin column, and place on the bench at RT for 15 min.
      Note: Add the Mix directly to the center of the RNeasy spin column membrane.
    10. Add 350 μl buffer RW1 to the RNeasy spin column. Close the lid gently and centrifuge at 8,000 x g for 15 sec. Discard the flow-through.
    11. Add 500 μl buffer RPE to the RNeasy spin column. Close the lid gently and centrifuge at 8,000 x g for 15 sec. Discard the flow-through.
    12. Add 500 μl buffer RPE to the RNeasy spin column. Close the lid gently and centrifuge at 8,000 x g for 2 min.
      Note: The long centrifugation dries the spin column membrane, eliminating ethanol contamination.
    13. Quickly remove the RNeasy spin column from the collection tube so that the column does not contact the flow through.
    14. Place the RNeasy spin column in a new 2 ml collection tube. Close the lid gently and centrifuge at full speed for 1 min.
      Note: It is important to eliminate any possible buffer RPE carryover.
    15. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30 μl RNase-free water or DEPC-water to the center of the spin column membrane. Incubate 1 min at RT. Close the lid gently and centrifuge at 8,000 x g for 1 min to elute the RNA.
    16. Add 20 μl RNase free water or DEPC-water to the center of spin column membrane to elute the maximum RNA. Incubate 1 min at RT. Close the lid gently and centrifuge at 8,000 x g for 1 min.
    17. Use Nanodrop and/or Bioanalyzer to test the RNA quantity and quality. You should get 5-10 μg total RNA from 250-300 collected galls at 3 dpi of a high quality.
    18. Store the RNA sample in -80 °C for future use.

Recipes

  1. High salt precipitation solution
    0.8 M sodium citrate
    1.2 M NaCl

Acknowledgments

This protocol is adapted from Barcala et al. (2010) and Portillo et al. (2013).

References

  1. Barcala, M., Garcia, A., Cabrera, J., Casson, S., Lindsey, K., Favery, B., Garcia-Casado, G., Solano, R., Fenoll, C. and Escobar, C. (2010). Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. Plant J 61(4): 698-712.
  2. Portillo, M., Cabrera, J., Lindsey, K., Topping, J., Andres, M. F., Emiliozzi, M., Oliveros, J. C., Garcia-Casado, G., Solano, R., Koltai, H., Resnick, N., Fenoll, C. and Escobar, C. (2013). Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression. New Phytol 197(4): 1276-1290.
  3. Portillo, M., Fenoll. C. and Escobar C. (2006). Evaluation of different RNA extraction methods for small quantities of plant tissue: Combined effects of reagent type and homogenisation procedure on RNA quality-integrity and yield. Physiol Plantarum 128 (1): 1-7.

简介

我们描述了一种有效的方法,以高质量和完整性从线虫诱导的gall中获得足够量的RNA,证明适合于转录组分析,即实时PCR,微阵列杂交或第二代测序 。 该方案对于少量胆汁(具有高蛋白质和糖含量的器官)是有效的。 该协议允许在感染后3天(dpi)从250-300个手部解剖的疱疹获得5-15μg总RNA的RNA产量(图1)。 它被证明特别适用于拟南芥和番茄。

关键字:瘿, 根结线虫, 高效RNA提取, transcriptomics, 高质量RNA


图1。 3 dpi 拟南芥 javanica 。蓝线表示收集的材料,加上一小部分根(容易处理),冷冻并加工以进行RNA提取。

材料和试剂

  1. M所感染的拟南芥。 javanica (3 dpi或7 dpi)
  2. TRI试剂(分子研究中心,MRC,目录号:TR-118)
  3. 氯仿(Scharlau,目录号:CL02031000)
  4. 异丙醇(Merck KGaA,目录号:09634)
  5. 柠檬酸钠(Sigma-Aldrich,目录号:s4641)
  6. 氯化钠(Duchefa,目录号:S0520)
  7. NaOH(Duchefa,目录号:s0523)
  8. 乙醇(Merck KGaA,目录号:100986)
  9. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758)
  10. 无RNase水
  11. 丙酮(Thermo Fisher Scientific,目录号:A/0600/17)
  12. 液氮
  13. RNeasy迷你包(QIAGEN,目录号:74104))
  14. RNase-Free DNase Set(QIAGEN,目录号:79254)
  15. 高盐沉淀溶液(见配方)

设备

  1. 离心机
  2. Microfuge
  3. 1.5 ml Eppendorf ®
  4. 瓷砂浆和杵,纹理表面在碗内部,直径60毫米
  5. 通风橱

程序

注意:对于所有程序,请使用手套和护目镜。

  1. 胆汁收集:为了以良好的效率进行从疱疹的RNA分离,应当收集在感染后3天(3dpi)或者在7dpi下100-150μg的250-300个手部切开的疱疹。 收集gall子,并在液氮中快速冷冻至1.5ml Eppendorf 管。
    注意:如果按比例缩小,RNA产量不与组织量成比例。 您可以在同一个Eppendorf ®中累积来自独立收集事件的。。

  2. 储存于-80℃直至进一步处理(步骤C)。

  3. 均质化:
    1. 用丙酮清洁砂浆和杵,让它们在工作台上干燥并高压灭菌以避免RNA酶活性。注意:检查提取过程中使用的砂浆,杵和移液管没有与RNases接触。有时,优选保持材料专用于RNA提取。
    2. 用杵将液氮加入研钵中冷却。
    3. 将galls加入砂浆,进入通风橱。
    4. 当液氮几乎蒸发时,加入TRI试剂(500-750μl/200-300加仑,3dpi);总体积不应超过用于均质化的TRI试剂的体积的10%,如TRI试剂 方案所示。
    5. 用研钵和杵研磨gall。 TRI试剂溶液最初变得冻结,但由于均质化它将逐渐融化。进行直到观察到液体外观,并且在试剂溶液内没有发现可见的组织碎片。
      注意:不要在没有均匀化的情况下留下痕迹,因为这一步可能会限制效率。
    6. 用移液管将胆汁匀浆转移到1.5ml Eppendorf管中,并将其置于冰上,同时使其余样品均匀。

  4. 相分离
    1. 在4℃下以12,000×g离心10分钟,并将含有RNA的上清液转移到新的1.5ml Eppendorf管中。
      注意:所得沉淀含有膜,多糖和高分子量DNA,而上清液含有RNA。 所有不溶物应从匀浆中除去。
    2. 在通风橱中每500μlTRl Reagent ®加入100μl氯仿。 牢固盖住样品管。
    3. 用手剧烈摇动管15秒,并在室温(RT)下孵育5至10分钟。
      注意:不要使用涡流。
    4. 在4℃下以12,000xg离心样品15分钟。
    5. 将上层水相转移到新管中
  5. RNA沉淀
    1. 每500μlTRl Reagent 加入125μl异丙醇和125μl高盐缓冲液。 混合溶液,在室温下保存5-10分钟。
      注意:高盐沉淀溶液可从分离的RNA中除去作为蛋白多糖和多糖的污染化合物。
    2. 在4℃下以12,000xg离心8分钟。
    3. 弃去上清液。

  6. RNA洗涤
    1. 用1ml 75%乙醇洗涤RNA沉淀,并颠倒混合数次
    2. 在4℃下以7,500×g离心5分钟,弃去上清液。
    3. 重复步骤E-1和2.
    4. 旋转10秒在微量离心机,仔细丢弃剩余的液体。
    5. 空气干燥颗粒3-5分钟。 不要让RNA过干,因为这会使其难以溶解。 白色RNA沉淀物在干燥时会变澄清。
    6. 在沉淀物清除后立即加入100μlDEPC-水或无RNase的水
    7. 通过在37℃下孵育10分钟,然后在60℃下5分钟溶解RNA
    8. 将RNA溶液储存在-80°C或继续进行RNA清洗通过DNase消化
  7. 在DNase I消化和RNA净化中合并。该程序基于两种不同的商业试剂盒(RNase-Free DNase Set和RNeasy Mini Kit)的组合使用。请按照其建议使用所有缓冲液和试剂的处理说明 注意:缓冲区作为RPE,RTL,RW1,RDD由套件中的公司提供。开始RNA清除之前,请确保DNA酶I和缓冲液RPE可以使用。
    1. DNase I储备溶液的制备:使用无RNase的针和注射器将550μl无RNA酶的水注入冻干的DNase I小瓶中。通过颠倒小瓶轻轻混合,并将其分成10μl一次性使用的等分试样。 -20℃保存9个月。
    2. 缓冲液RPE的制备:如瓶中所示加入4体积的96-100%乙醇(44ml)
    3. 用无RNase的水将RNA样品体积调节至100μl。
    4. 加入350μl缓冲液RLT,并通过吸移混匀
    5. 加入250μlEtOH,通过吸移混匀
    6. 将样品转移到置于2ml收集管中的RNeasy Mini离心柱。轻轻关闭盖子,以8,000×g离心15秒。丢弃流出物。
    7. 向RNeasy离心柱中加入350μl缓冲液RW1。轻轻关闭盖子,以8,000×g离心15秒。丢弃流出物。
    8. 加入70μl缓冲液RDD(随RNase-Free DNase Set提供)至10μlDNA酶I储备液。通过倒置管子轻轻混合。
      注意:不要涡旋。
    9. 将80μlDNase I孵育混合物加入RNeasy离心柱中,在室温下置于台上15分钟。
      注意:将混合物直接添加到RNeasy旋转柱膜的中心。
    10. 向RNeasy离心柱中加入350μl缓冲液RW1。轻轻关闭盖子,以8,000×g离心15秒。丢弃流出物。
    11. 向RNeasy离心柱中加入500μl缓冲液RPE。轻轻关闭盖子,以8,000×g离心15秒。丢弃流出物。
    12. 向RNeasy离心柱中加入500μl缓冲液RPE。轻轻关闭盖子并以8,000×g离心2分钟。
      注意:长离心干燥旋转柱膜,消除乙醇污染。
    13. 从收集管中快速取出RNeasy离心柱,以使色谱柱不接触流出物
    14. 将RNeasy离心柱置于新的2ml收集管中。轻轻关闭盖子,全速离心1分钟。
      注意:重要的是消除任何可能的缓冲区RPE残留。
    15. 将RNeasy离心柱置于新的1.5 ml收集管中。加入30μl无RNase水或DEPC水到离心柱膜的中心。在室温孵育1分钟。轻轻关闭盖子并以8,000×g离心1分钟以洗脱RNA。
    16. 添加20微升无RNA酶水或DEPC水到离心柱膜的中心,以洗脱最大RNA。 在室温孵育1分钟。 轻轻关闭盖子,以8000xg离心1分钟。
    17. 使用Nanodrop和/或生物分析仪测试RNA的数量和质量。 你应该得到5-10微克的总RNA,从250-300收集的gall,在3 dpi的高质量
    18. 将RNA样品储存在-80℃以备将来使用

食谱

  1. 高盐沉淀溶液
    0.8 M柠檬酸钠 1.2 M NaCl

致谢

该协议改编自Barcala等人(2010)和Portillo等人(2013)。

参考文献

  1. Barcala,M.,Garcia,A.,Cabrera,J.,Casson,S.,Lindsey,K.,Favery,B.,Garcia-Casado,G.,Solano,R.,Fenoll,C.and Escobar,C 。(2010)。 显微解剖的拟南芥线虫诱导的巨细胞中的早期转录组事件。 a> Plant J 61(4):698-712。
  2. Portillo,M.,Cabrera,J.,Lindsey,K.,Topping,J.,Andres,MF,Emiliozzi,M.,Oliveros,JC,Garcia-Casado,G.,Solano,R.,Koltai, Resnick,N.,Fenoll,C。和Escobar,C。(2013)。 番茄相比拟南芥中的线虫诱导的巨细胞发育过程中的不同和保守的转录组变化:功能角色 用于基因抑制。新植物 197(4):1276-1290。
  3. Portillo,M.,Fenoll。 C.和Escobar C.(2006)。 评估少量植物组织的不同RNA提取方法: 试剂类型和均质化程序对RNA质量完整性和产量的组合效应。 生理药理学 128(1):1-7。
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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:García, A., Barcala, M., Cabrera, J., Fenoll, C. and Escobar, C. (2013). RNA Isolation From Meloidogyne Spp. Galls. Bio-protocol 3(17): e879. DOI: 10.21769/BioProtoc.879.
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