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Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
从葡萄果皮上提取总RNA进行定量反转录PCR和基因芯片分析   

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参见作者原研究论文

本实验方案简略版
Plant Physiology
May 2015

Abstract

Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).

Keywords: RNA extraction (RNA的提取), Grape berry skin (葡萄浆果皮), Hot Borate Method (热硼酸法)

Materials and Reagents

  1. 50 ml centrifuge tubes (e.g., the Falcon tubes)
  2. 1.5 ml microcentrifuge tubes
  3. 2 ml microcentrifuge tubes
  4. Stainless steel spoon
  5. Grape berry skins
    Notes:
    1. Skins isolated from grape berries using tweezers (Figure 1A, Video 1) . Skins were put in 15 or 50 ml tubes and frozen in liquid nitrogen immediately (Figure 1B, Video 1). These were stored at -80 °C. We recommend that preparation of about 20 pieces (about 1 g skin) because there are variations among berries.
    2. This method could be applied to all grape varieties.
  6. Liquid nitrogen
  7. Proteinase K solutions (20 mg/ml) (Siyaku, Wako Pure Chemical Industries, catalog number: 160-22752 )
  8. Lithium chloride (LiCl) (2 M, 10 M) (Siyaku, Wako Pure Chemical Industries, catalog number: 127-01165 )
    Note: DEPC treated and autoclaved.
  9. 2 M potassium acetate (KOAC) (Siyaku, Wako Pure Chemical Industries, catalog number: 160-03175 )
    Note: DEPC treated and autoclaved.
  10. 10 mM Tris-HCl (pH 7.5) (Siyaku, Wako Pure Chemical Industries, catalog number: 207-06275 )
    Note: DEPC treated and autoclaved.
  11. 1 M potassium chloride (KCl) (Siyaku, Wako Pure Chemical Industries, catalog number: 163-03545 )
    Note: DEPC treated and autoclaved.
  12. Ethanol [70%, 99.8% (v/v)] (Siyaku, Wako Pure Chemical Industries, catalog number: 057-00456 )
  13. RNeasy Plant Mini Kit (QIAGEN, catalog number: 74903 or 74904 )
  14. RNase free water
  15. Sodium borate decahydrate (Borax) (Siyaku, Wako Pure Chemical Industries, catalog number: 194-01415 )
  16. Ethylene glycol tetraacetic acid (EGTA) (Dojindo, catalog number: 346-01312 )
  17. 1% (w/v) sodium dodecyl sulfate (SDS) (Siyaku, Wako Pure Chemical Industries, catalog number: 191-07145 )
  18. 1% (w/v) deoxycholate sodium salt (Siyaku, Wako Pure Chemical Industries, catalog number: 192-08312 )
  19. 10 mM dithiothreitol (DTT) (Siyaku, Wako Pure Chemical Industries, catalog number: 045-08974 )
  20. 1% (w/v) Triton X-114 (Sigma-Aldrich, catalog number: X114 )
  21. 2% (w/v) polyvinylpyrrolidone (PVP-40) (Sigma-Aldrich, catalog number: PVP40 )
  22. Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 ) treated and autoclaved
  23. Hot Borate Extraction Buffer (see Recipes)

Equipment

  1. Crusher (Automill) (Tokken, model: TK-AM5-H )
  2. Shaking water bath (42 °C) (TAITEC CORPORATION, model: THERMO MINDER SM-05 with PERSONAL-11)
  3. Centrifuge with angle rotor for 50 ml tube (SAKURA, model: 50A-7 with 50F-8A)
  4. Centrifuge with angle rotor for 1.5 ml tube (SAKURA, model: SS1500X with 15M-24AM)
  5. Vacuum equipment (EYELA, model: CVE-2000 )

Procedure

  1. Extraction of RNA from the sample
    1. Grape berry skins are prepared as shown in Figure 1 and Video 1.


      Figure 1. Preparation of grape berry skins. Grape skins were separated from berry (A) and frozen in liquid nitrogen (B)

      Video 1. Preparation of grape berry skins

    2. Powder frozen grape berry skins with liquid N2 using the crusher (1,300 rpm, 30 sec) (Figure 2A-B, Video 2).


      Figure 2. The crusher (A) and powdered grape berry skins (B)

      Video 2. Powder frozen grape berry skins with liquid N2 using the crusher

    3. Put 0.2 g powdered tissue into 50 ml centrifuge tube using the cold spoon (Figure 3A, Video 3).


      Figure 3. Preparation of RNA sample. A. Powdered tissue was put into tube using the cold spoon; B. The sample was mixed by vortexing.

      Video 3. Preparation of RNA sample

    4. Add 2 ml hot borate extraction buffer (42 °C) (Video 3).
    5. Add 10 μl Proteinase K solutions (20 mg/ml) immediately (Video 3).
    6. Mix the sample by vortexing until well mixed (approximately 20 sec) (Figure 3B, Video 3).
    7. Incubate the tube at 42 °C in the shaking water bath for 1.5 h.
    8. Centrifuge at 8,000 rpm (8,000 x g) for 10 min at room temperature.
    9. Transfer the supernatant to three 1.5 ml tubes (600 μl/tube).
    10. Add 114.3 μl of 1 M KCl to each tube to adjust a final concentration of 160 mM.
    11. Incubate the tubes on ice for 1 h.
    12. Centrifuge at 10,000 rpm (12,000 x g) for 30 min at 4 °C to remove debris.
    13. Transfer the supernatants to 1.5 ml microcentrifuge tube individually.
    14. Discard pellet and use transferred supernatant in step B (see below).

  2. Purification of RNA (remove polysaccharide)
    1. Add 178.5 μl of 10 M LiCl to each tube to adjust a final concentration of 2 M.
    2. Incubate on ice in a cold room overnight.
    3. Centrifuge at 10,000 rpm (12,000 x g) for 30 min at 4 °C.
    4. Decant and discard the supernatant immediately.
    5. Add 200 μl of cold 2 M LiCl for each tube.
    6. Suspend the pellet by vortexing until well mixed (approximately 20 sec).
    7. Centrifuge at 10,000 rpm (12,000 x g) for 10 min at 4 °C.
    8. Decant and discard the supernatant immediately.
    9. Repeat steps B5-8 twice (for a total of three times).
    10. If the pellet contains pigment, repeat steps B5-8 until the pellet becomes colorless.
    11. Suspend the pellet in 100 μl of 10 mM Tris-HCl (pH 7.5) at room temperature.
    12. Collect the suspensions in one 1.5 ml microcentrifuge tube and adjust to 400 μl with 10 mM Tris-HCl (pH 7.5).
    13. Centrifuge at 10,000 rpm (12,000 x g) for 10 min at 4 °C to remove insoluble materials.
    14. Transfer the supernatant to 1.5 ml microcentrifuge tube.
    15. Add 40 μl of 2 M KOAC to adjust a final concentration of 0.2 M.
    16. Incubate on ice for 15 min.
    17. Centrifuge at 10,000 rpm (12,000 x g) for 10 min at 4 °C.
    18. Transfer the supernatant to 2 ml microcentrifuge tube.
    19. Add 1.1 ml of 99.8% ethanol and keep at -20 °C overnight to precipitate RNA.

  3. Cleanup and concentration of RNA
    1. Centrifuge at 10,000 rpm (12,000 x g) for 30 min at 4 °C to pellet RNA.
    2. Decant and discard ethanol.
    3. Wash pellet with 70% (v/v) ethanol.
    4. Centrifuge at 10,000 rpm (12,000 x g) for 10 min at 4 °C.
    5. Decant and discard ethanol.
    6. Remove residual ethanol under vacuum.
    7. Suspend the pellet in 100 μl RNase free water.
    8. Stored at -80 °C until use for qRT-PCR or keep at 4 °C until further purification.

  4. Further cleanup and concentration of RNA for microarray analysis
    1. The RNA was further purified using RNeasy Plant Mini Kit according to the supplier’s protocol.
    2. Add 350 μl of RLT buffer and mix thoroughly.
    3. Add 250 μl of 99.8% ethanol and mix well by pipetting.
    4. Apply sample to RNeasy mini spin column sitting with collection tube.
    5. Centrifuge at 8,000 rpm (8,000 x g) for 15 sec.
    6. Transfer the column to a new tube.
    7. Add 500 μl of RPE buffer and centrifuge at 8,000 rpm (8,000 x g) for 15 sec.
    8. Add 500 μl of RPE buffer and centrifuge at 8,000 rpm (8,000 x g) for 2 min.
    9. Discard flow-through and transfer the column to a new tube.
    10. Centrifuge at 10,000 rpm (12,000 x g) for 1 min.
    11. Discard flow-through and transfer the column to a new tube.
    12. Add 100 μl of water and centrifuge at 8,000 rpm (8,000 x g) for 1 min.
    13. Add 100 μl of water and centrifuge at 8,000 rpm (8,000 x g) for 1 min.
    14. Collect the filtrates and add 500 μl of 99.8% ethanol.
    15. Keep at -20 °C overnight to precipitate RNA.
    16. Repeat steps C1-6.
    17. Suspend the pellet in 10 μl RNase free water.
    18. Stored at -80 °C until use for microarray analysis.

Recipes

  1. Hot borate extraction buffer
    0.2 M sodium borate decahydrate (Borax)
    30 mM ethylene glycol tetraacetic acid (EGTA)
    1% (w/v) sodium dodecyl sulfate (SDS)
    1% (w/v) deoxycholate sodium salt
    10 mM dithiothreitol (DTT)
    1% (w/v) Triton X-114
    2% (w/v) polyvinylpyrrilidone (PVP-40)
    Notes:
    1. Store Borax, EGTA, SDS and deoxycholate sodium salt as 1.5x mixture after autoclave.
    2. DTT was filtered and stored as 1 M solution at -80 °C.
    3. Store PVP-40 as 20% (w/v) solution after autoclave.
    4. Add DTT, Triton X-114 and PVP-40 before use.

Acknowledgments

This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry from the Bio-oriented Technology Research Advancement Institution (BRAIN) and by Grants-in-Aid for Scientific Research from The Japan Society for the Promotion of Science (JSPS).
We thank Dr. Wan and Dr. Wilkins for showing method of ‘Hot Borate Method’.
We also thank Dr. Azuma and Mr. Nakao for helping us making of figures and videos.

References

  1. Suzuki, M., Jasinski, M., Martinoia, E., Nakabayashi, R., Suzuki, M., Saito, K. and Shiratake, K. (2015a). Molecular cloning and characterization of ABCG/PDRtype ABC transporter in grape berry skin. Adv Hortic Sci 28: 53-63.
  2. Suzuki, M., Nakabayashi, R., Ogata, Y., Sakurai, N., Tokimatsu, T., Goto, S., Suzuki, M., Jasinski, M., Martinoia, E., Otagaki, S., Matsumoto, S., Saito, K. and Shiratake, K. (2015b). Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation. Plant Physiol 168(1): 47-59.
  3. Wan, C. Y. and Wilkins, T. A. (1994). A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223(1): 7-12.

简介

高质量RNA的提取是定量逆转录PCR(qRT-PCR)和微阵列分析的必要步骤。 然而,从含有大量多糖,脂质和次级代谢物的水果材料中提取高质量RNA是不容易的。 Wan和Wilkins(1994)开发了"热硼酸盐方法"来分离高质量的RNA。 在这里,我们描述了"热硼酸盐方法"的修饰的协议,从葡萄浆果皮肤中分离高质量的RNA用于qRT-PCR和微阵列分析(Suzuki等人,2015a; Suzuki等人 al。,2015b)。

关键字:RNA的提取, 葡萄浆果皮, 热硼酸法

材料和试剂

  1. 50ml离心管(例如 Falcon管)
  2. 1.5 ml微量离心管
  3. 2 ml微量离心管
  4. 不锈钢勺
  5. 葡萄浆果皮
    注意:
    1. 使用镊子从葡萄浆果中分离的皮肤(图1A,视频1)。 将皮肤置于15或50ml管中并在液氮中冷冻 立即(图1B,视频1)。将它们储存在-80℃。我们 推荐准备约20片(约1克皮肤)因为 浆果中有变化。
    2. 此方法可应用于所有葡萄品种。
  6. 液氮
  7. 蛋白酶K溶液(20mg/ml)(Siyaku,Wako Pure Chemical Industries,目录号:160-22752)
  8. 氯化锂(LiCl)(2M,10M)(Siyaku,Wako Pure Chemical Industries,目录号:127-01165)
    注意:DEPC处理和高压灭菌。
  9. 2 M乙酸钾(KOAC)(Siyaku,Wako Pure Chemical Industries,目录号:160-03175)
    注意:DEPC处理和高压灭菌。
  10. 10mM Tris-HCl(pH7.5)(Siyaku,Wako Pure Chemical Industries,目录号:207-06275)
    注意:DEPC处理和高压灭菌。
  11. 1M氯化钾(KCl)(Siyaku,Wako Pure Chemical Industries,目录号:163-03545)
    注意:DEPC处理和高压灭菌。
  12. 乙醇[70%,99.8%(v/v)](Siyaku,Wako Pure Chemical Industries,目录号:057-00456)
  13. RNeasy Plant Mini Kit(QIAGEN,目录号:74903或74904)
  14. 无RNase水
  15. 硼酸钠十水合物(硼砂)(Siyaku,Wako Pure Chemical Industries,目录号:194-01415)
  16. 乙二醇四乙酸(EGTA)(Dojindo,目录号:346-01312)
  17. 1%(w/v)十二烷基硫酸钠(SDS)(Siyaku,Wako Pure Chemical Industries,目录号:191-07145)
  18. 1%(w/v)脱氧胆酸钠盐(Siyaku,Wako Pure Chemical Industries,目录号:192-08312)
  19. 10mM二硫苏糖醇(DTT)(Siyaku,Wako Pure Chemical Industries,目录号:045-08974)
  20. 1%(w/v)Triton X-114(Sigma-Aldrich,目录号:X114)
  21. 2%(w/v)聚乙烯吡咯烷酮(PVP-40)(Sigma-Aldrich,目录号:PVP40)
  22. 重复处理并高压灭菌的焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758)
  23. 热硼酸盐提取缓冲液(参见配方)

设备

  1. 破碎机(自动机)(Tokken,型号:TK-AM5-H)
  2. 摇动水浴(42℃)(TAITEC CORPORATION,型号:带有PERSONAL-11的THERMO MINDER SM-05)
  3. 用角度转子离心50ml管(SAKURA,型号:50A-7,用50F-8A)
  4. 用角度转子离心1.5ml管(SAKURA,型号:SS1500X,含15M-24AM)
  5. 真空设备(EYELA,型号:CVE??-2000)

程序

  1. 从样品中提取RNA
    1. 如图1和视频1所示准备葡萄浆果皮。


      图1.制备葡萄浆果皮。将葡萄皮与浆果(A)分离,在液氮(B)中冷冻

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    2. 使用破碎机(1,300rpm,30秒)(图2A-B,视频2),用液体N 2粉末冷冻的葡萄浆果皮。


      图2.破碎机(A)和粉状葡萄浆果皮(B)

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      视频2.使用破碎机的粉末冷冻的葡萄浆果皮肤与液体N
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    3. 使用冷勺将0.2 g粉末组织放入50 ml离心管中(图3A,视频3)

      图3. RNA样品的制备。A.将粉末状组织放入 管用冷勺; B.通过涡旋混合样品。

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      视频3.准备RNA样本
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    4. 加入2ml热硼酸盐萃取缓冲液(42℃)(视频3)
    5. 立即加入10μl蛋白酶K溶液(20 mg/ml)(视频3)
    6. 通过涡旋混合样品,直到充分混合(约20秒)(图3B,视频3)
    7. 将管在42℃下在振荡水浴中孵育1.5小时
    8. 在室温下以8,000rpm(8,000xg)离心10分钟。
    9. 将上清转移到三个1.5ml管(600μl/管)
    10. 向每个管中加入114.3μl1M KCl以调节终浓度为160mM
    11. 在冰上孵育管1小时。
    12. 在4℃下以10,000rpm(12,000xg)离心30分钟以除去碎屑。
    13. 将上清液单独转移到1.5ml微量离心管中
    14. 弃去沉淀并在步骤B中使用转移的上清液(见下文)。

  2. RNA纯化(去除多糖)
    1. 向每个管中加入178.5μl10M LiCl以调节终浓度为2M
    2. 在冷室中在冰上孵育过夜。
    3. 在4℃下以10,000rpm(12,000xg)离心30分钟。
    4. 立即倾析并弃去上清液。
    5. 每管加入200μl冷的2M LiCl
    6. 通过涡旋悬浮颗粒,直到充分混合(约20秒)
    7. 在4℃下以10,000rpm(12,000xg)离心10分钟。
    8. 立即倾析并弃去上清液。
    9. 重复步骤B5-8两次(共三次)。
    10. 如果颗粒含有颜料,重复步骤B5-8直到颗粒变为无色
    11. 在室温下将沉淀悬浮在100μl10mM Tris-HCl(pH7.5)中
    12. 收集悬浮液在一个1.5毫升微量离心管,并用10毫米Tris-HCl(pH 7.5)调整到400微升。
    13. 在4℃下以10,000rpm(12,000xg)离心10分钟以除去不溶性物质。
    14. 将上清液转移到1.5ml微量离心管中
    15. 加入40μl的2M KOAC调节终浓度0.2μM
    16. 在冰上孵育15分钟。
    17. 在4℃下以10,000rpm(12,000xg)离心10分钟。
    18. 将上清液转移到2ml微量离心管中
    19. 加入1.1ml的99.8%乙醇,并保持在-20℃过夜以沉淀RNA。

  3. RNA净化和浓缩
    1. 在4℃下以10,000rpm(12,000xg)离心30分钟以沉淀RNA。
    2. 滗析和丢弃乙醇。
    3. 用70%(v/v)乙醇洗涤沉淀
    4. 在4℃下以10,000rpm(12,000xg)离心10分钟。
    5. 滗析和丢弃乙醇。
    6. 在真空下除去残留的乙醇。
    7. 将沉淀悬浮在100μl无RNA酶的水中
    8. 储存在-80°C,直到用于qRT-PCR或保持在4°C直到进一步纯化。

  4. 进一步净化和浓缩RNA用于微阵列分析
    1. 使用RNeasy Plant Mini Kit根据供应商的方案进一步纯化RNA
    2. 加入350μlRLT缓冲液,彻底混匀
    3. 加入250微升99.8%乙醇,通过吸移混匀
    4. 将样品应用于装有收集管的RNeasy微型离心柱
    5. 以8,000rpm(8,000×g)离心15秒
    6. 在新管上转移色谱柱。
    7. 加入500μlRPE缓冲液,并以8,000rpm(8,000×g )离心15秒。
    8. 加入500μlRPE缓冲液,并以8,000rpm(8,000×g )离心2分钟。
    9. 弃去流出物并在新管上转移色谱柱。
    10. 以10,000rpm(12,000xg)离心1分钟。
    11. 弃去流出物并在新管上转移色谱柱。
    12. 加入100μl水并以8,000rpm(8,000×g )离心1分钟。
    13. 加入100μl水并以8,000rpm(8,000×g )离心1分钟。
    14. 收集滤液,加入500μl99.8%乙醇
    15. 保持在-20°C过夜以沉淀RNA
    16. 重复步骤C1-6。
    17. 将沉淀悬浮在10μl无RNA酶的水中。
    18. 储存于-80℃直至用于微阵列分析。

食谱

  1. 热硼酸盐萃取缓冲液
    0.2M硼酸钠十水合物(Borax)
    30mM乙二醇四乙酸(EGTA)
    1%(w/v)十二烷基硫酸钠(SDS)
    1%(w/v)脱氧胆酸钠盐 10mM二硫苏糖醇(DTT) 1%(w/v)Triton X-114 2%(w/v)聚乙烯吡咯烷酮(PVP-40) 注意:
    1. 高压灭菌后,将硼砂,EGTA,SDS和脱氧胆酸钠盐以1.5x混合物储存。
    2. 过滤DTT并在-80℃下以1M溶液储存。
    3. 高压灭菌后将PVP-40作为20%(w/v)溶液贮存。
    4. 在使用前添加DTT,Triton X-114和PVP-40。

致谢

这项工作得到了来自生物技术研究推进机构(BRAIN)的促进生物工业创新的基础和应用研究计划以及来自日本促进学会的科学研究助学金of Science(JSPS)。
我们感谢Wan博士和Wilkins博士的"热硼酸盐法"的显示方法。
我们也感谢Azuma博士和Nakao先生帮助我们制作数字和视频。

参考文献

  1. Suzuki,M.,Jasinski,M.,Martinoia,E.,Nakabayashi,R.,Suzuki,M.,Saito,K.and Shiratake,K。(2015a)。 ABCG/PDRtype ABC转运蛋白在葡萄浆果皮中的分子克隆和表征 Adv Hortic Sc??i 28:53-63。
  2. Suzuki,M.,Nakabayashi,R.,Ogata,Y.,Sakurai,N.,Tokimatsu,T.,Goto,S.,Suzuki,M.,Jasinski,M.,Martinoia,E.,Otagaki, Matsumoto,S.,Saito,K。和Shiratake,K。(2015b)。 葡萄浆果皮肤中的多元学显示紫外线-C辐射对芪合成途径的特异性诱导。 a> 植物生理学 168(1):47-59。
  3. Wan,C.Y。和Wilkins,T.A。(1994)。 改良的热硼酸盐方法显着提高了来自棉花的高质量RNA的产量( Gossypium hirsut L.)。 Anal Biochem 223(1):7-12。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Suzuki, M. and Shiratake, K. (2016). Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis. Bio-protocol 6(7): e1777. DOI: 10.21769/BioProtoc.1777.
  2. Suzuki, M., Nakabayashi, R., Ogata, Y., Sakurai, N., Tokimatsu, T., Goto, S., Suzuki, M., Jasinski, M., Martinoia, E., Otagaki, S., Matsumoto, S., Saito, K. and Shiratake, K. (2015b). Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation. Plant Physiol 168(1): 47-59.
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