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Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small RNAs have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Northern blot analysis of small RNAs involving the separation of RNA molecules using polyacrylamide gel electrophoresis (PAGE) has remained a popular and valuable analytical method to validate small RNAs. Northern blot analysis consist of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization using radioactive probes. The following protocol provides a method for isolation and detection of small RNAs from virus-infected plants and was successfully used in Panwar et al. (2013a), Panwar et al. (2013b).

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A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants
高通量检测病毒感染植株中siRNA

植物科学 > 植物分子生物学 > RNA > RNA 干扰
作者: Vinay Panwar
Vinay PanwarAffiliation: Plant Biotechnology Institute, National Research Council Canada, Saskatoon, Saskatchewan, Canada
For correspondence: vinay.panwar@nrc-cnrc.gc.ca
Bio-protocol author page: a918
 and Guus Bakkeren
Guus BakkerenAffiliation: Pacific Agri-Food Research Centre, Agriculture & Agri-Food Canada, Summerland, Canada
For correspondence: guus.bakkeren@agr.gc.ca
Bio-protocol author page: a919
Vol 3, Iss 20, 10/20/2013, 4553 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.940

[Abstract] Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small RNAs have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Northern blot analysis of small RNAs involving the separation of RNA molecules using polyacrylamide gel electrophoresis (PAGE) has remained a popular and valuable analytical method to validate small RNAs. Northern blot analysis consist of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization using radioactive probes. The following protocol provides a method for isolation and detection of small RNAs from virus-infected plants and was successfully used in Panwar et al. (2013a), Panwar et al. (2013b).
Keywords: RNA Silencing(RNA沉默), SiRNA Detection(基因检测), Northern Blotting(Northern印迹法), Virus-Induced Gene Silencing(病毒诱导的基因沉默), Host-Induced Gene Silencing(宿主诱导的基因沉默)

[Abstract]

Materials and Reagents

  1. Virus-infected plant tissue
  2. TRIzol reagent (Invitrogen)
  3. Chloroform (Sigma-Aldrich)
  4. Isopropanol (Sigma-Aldrich)
  5. Ethyl alcohol (EtOH)
  6. Diethylpyrocarbonate (DEPC) (Sigma-Aldrich)
  7. Hybond NX Neutral Membrane (Amersham biosciences, catalog number: RPN203T )
  8. 40% Acrylamide/N’N’-bis-methylene-acrylamide (19:1) (Life Technologies, Ambion®)
  9. Tetramethylethylenediamine (EDTA) (Sigma-Aldrich)
  10. Urea (Sigma-Aldrich)
  11. Hyperfilm TM MP (Amersham biosciences, catalog number: 28-9068-45 )
  12. Ethidium bromide (Sigma-Aldrich)
  13. Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma-Aldrich, catalog number: 39391 )
  14. Ammonium persulphate (APS)
  15. Tetramethylethylenediamine (TEMED) (Sigma-Aldrich)
  16. Tris base (AMRESCO)
  17. Boric acid (Fisher Scientific)
  18. Formamide (Sigma-Aldrich)
  19. Bromophenol blue (Sigma-Aldrich)
  20. Xylene cyanol (Sigma-Aldrich)
  21. 3 MM Whatman filter paper
  22. 1-methylimidazole (Sigma-Aldrich)
  23. Hydrochloric acid (HCl)
  24. Sodium dodecyl sulphate (SDS) (Fisher Scientific)
  25. 20x Saline sodium citrate (SSC) buffer (Sigma-Aldrich)
  26. ULTRAhyb-Oligo buffer (Life Technologies, Ambion®, catalog number: AM8669 )
  27. Megaprime DNA Labeling System (General Electric Company, model: RPN1604 )
  28. Liquid nitrogen
  29. RNase-free water
  30. Small gels (size of regular protein gels 1.5 mm thick) (Bio-Rad, Mini-Protean® Cell)
  31. Neutral Hybond nitrocellulose membrane
  32. RNase ZAP (Life Technologies, catalog number: AM9784 )
  33. DEPC treated water (see Recipes)
  34. 10x Tris-Borate-EDTA (TBE) buffer (see Recipes)
  35. 10% APS (see Recipes)
  36. 15% polyacrylamide gel (see Recipes)
  37. 2x loading buffer (see Recipes)

Equipment

  1. Pestle and mortars
  2. Semi dry electroblotter (Bio-Rad)
  3. Protean II vertical gel system (Bio-Rad)
  4. Nanodrop spectrophotometer
  5. Centrifuge
  6. Hybridization oven
  7. UV transilluminator
  8. Microcentrifuge tube
  9. Electrophoresis apparatus
  10. X-ray film

Procedure

  1. RNA isolation
    1. Using mortar and pestle grind 50-100 mg of virus-infected plant tissue in liquid nitrogen. Immediately add 1 ml of TRIzol reagent and continue grinding until a uniform paste is formed. Pipet the homogenized material in to a sterile microcentrifuge tube and incubate for 5 min at 25-30 °C to permit complete dissociation of nucleoprotein complexes.
    2. Add 200 μl of chloroform per ml of TRIzol reagent used for homogenization of sample tissue. Shake the samples vigorously by inverting the microcentrifuge tubes several times by hand and incubate at 20-30 °C for 2-3 min.
    3. Centrifuge the samples at no more than 12,000 x g for 15 min at room temperature (20-25 °C).
    4. Following centrifugation the mixture will separate in a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase.
    5. Without disturbing the interphase, carefully pipette out the aqueous phase and transfer it to a new sterile microcentrifuge tube.
    6. Precipitate the RNA from the aqueous phase by adding 0.5 ml of isopropanol per ml of TRIzol reagent used.
    7. Incubate sample at 20-30 °C for 10 min. Recover the total RNA by centrifuging the sample at no more than 12,000 x g for 10 min at 2-8 °C. RNA will form a gel like pellet at the bottom and side of the microcentrifuge tube.
    8. Without disturbing the pellet remove the supernatant. Wash the RNA pellet by slowly adding 1 ml of 75% ethanol down the side of the tube.
    9. Mix the sample briefly by vortexing and centrifuge at no more than 7,500 x g for 5 min at 2-8 °C.
    10. Discard the supernatant carefully without disturbing the pellet. Vacuum or air dry the pellet for 10-15 min and resuspend in 50 μl RNase-free water (commercially available or DEPC-treated sterile water). The volume is adjustable but higher concentrations are preferable since this will allow lower volumes to be loaded on the gel. Do not overdry the pellet otherwise it will be hard to dissolve. Quantify the concentration of RNA by measuring the absorbance at 260 nm using a spectrophotometer (e.g., Nanodrop) and store at -20 °C until ready to use. RNA will be stable for months at these conditions.

  2. 15% Polyacrylamide gel electrophoresis (PAGE)
    Small gels (size of regular protein gels 1.5 mm thick) are good to detect the expression of small RNAs. However, to analyze length variants of individual small RNAs (e.g. 20 nt, 21 nt, 22 nt), you need to run longer gels. Before assembling the electrophoresis system, clean all equipment’s thoroughly for any RNase contamination using RNase ZAP. Avoid nuclease contamination throughout the procedure by using sterile solutions and RNase free plasticware.
    1. Assemble gel casting chamber following manufacturer instructions and handcast 15% polyacrylamide gel. Let the gel polymerize for at least 30 min to 1 h at room temperature (20-25 °C). Setup the electrophoresis module as recommended by the manufacturer. Rinse the wells thoroughly with running buffer (0.5x TBE) using a syringe to remove any traces of urea and pre run gel with tracking dye for nearly 15 min before loading the samples.
    2. While pre-running the gel, prepare RNA samples. Ideally, for a good siRNAs resolution, 35-40 μg of total RNA is recommended for loading (based on the purity and concentration of the RNA). Accordingly, adjust the volume of each sample to be loaded (according to the least concentrated sample) using DEPC-treated water. Add equal volumes of 2x loading dye to the samples. Mix well by gentle tapping and spin down briefly.
    3. Cap the tubes and denature RNA samples by heating at 90-100 °C for 15 min. Spin down the samples briefly followed by snap cooling on ice for 5 min.
    4. Load the samples and the siRNA size marker and run the gel at 200 V for nearly an hour.
      Note: The amount of voltage and duration of the run for the gel depends on the types of power supply and gel-electrophoresis system used. Stop the run when the bromophenol blue dye has migrated to the end of the gel. Do not overrun.

  3. Semi-dry electroblotting of siRNAs onto nylon membrane
    1. Carefully dismantle the electrophoresis apparatus without breaking the gel. Cut the lower portion of the gel containing both tracking dye (xylene cyanol and bromophenol blue) bands. On a 15% polyacrylamide gel, the bromophenol blue dye front runs near 10 nt long RNA. The upper portion of the gel which includes tRNA and 5S RNA is stained with ethidium bromide solution (0.25 μg/ml ethidium bromide in 0.5x TBE buffer) for 10-20 min. Inspect the integrity and equal loading of RNA in the gel using a 360 nm UV transilluminator. The sharpness and clarity of tRNA bands is a good indicator of RNA quality and integrity (Figure 1).


      Figure 1. Northern blot revealing small interfering RNA molecules of 23 nucleotides (upper panel). Ethidium bromide-stained rRNA served as loading controls in the gel prior to RNA transfer (lower panel).

    2. To prepare a transfer sandwich, cut 3 MM Whatman filter paper (six pieces) and neutral Hybond nitrocellulose membrane to the size of the gel. Soak the membrane and Whatman paper in 0.5x TBE buffer until they are wet completely. Complete wetting of the membrane is important to insure proper binding of nucleic acids. Assemble the sandwich as follows.
    3. Place three pieces of Whatman paper on the anode plate of electroblotter. Make sure to roll out any air bubble that may inhibit transfer.
    4. Transfer the membrane on top of the filter papers and roll out any air bubble formed between the membrane and filer paper. After electrophoresis, equilibrate the gel in transfer buffer (0.5x TBE) for 5 to 10 min. Equilibration facilitates the removal of electrophoresis buffer salts and detergents. Carefully place the equilibrated gel over the membrane, aligning the stack as perfect as possible.
    5. Place the remaining three pieces of Whatman paper saturated in running buffer on the stack and roll out any trapped air.
    6. Slightly wet the stack with transfer buffer (0.5x TBE) and place the anode plate on the top without disturbing the gel-nitrocellulose stack and secure firmly.
      Note: It is important to exclude excess buffer and air bubbles trapped in the filter paper and membrane when setting up the transfer.
    7. Set the power supply and run the transfer unit for 30 min at 10 V and 200 mAmp (constant current settings). Carefully disassemble the transfer unit and remove the filter paper and gel on top of the nylon membrane. Mark the orientation of the gel slots on the membrane with a pencil on RNA transfer side.

  4. Chemical cross linking
    Cross linking of the RNA to the membrane frequently improves the sensitivity of northern blots. RNA can be immobilized to the membrane using conventional methods such as exposure to standard dose of UV (120 mJ/cm2) or by baking in an oven (80 °C for 30 min). Both of these methods can be used successfully, however using the EDC-based cross-linking of small RNAs to membrane greatly improves the signal resolution of small RNAs by hybridization (Pall and Hamilton, 2008).
    1. Immediately prior to use, prepare a solution of 0.16 M EDC solution in 0.13 M 1-methylimidazole and adjust the pH of the solution to 8 with HCl.
    2. Cut a single sheet of 3 MM Whatman filter paper slightly bigger than the size of the membrane and saturate it with the EDC solution.
    3. The membrane is placed on the EDC-saturated 3 MM paper with the side on to which RNA was transferred facing up. Roll out any air bubbles trapped between the membrane and filter paper.
    4. Cover the tray holding the membrane and filter paper with saran wrap and incubate at 60 °C for between 1 to 2 h.
    5. Remove the membrane and rinse with RNase free water to remove any residual EDC solution.
    6. The membrane can be dried and stored at -20 °C after removal of residual EDC without compromising the sensitivity of siRNA detection, or be used immediately for hybridization.

  5. Hybridization
    Finally, the membranes with fixed RNAs are incubated with specific, radiolabelled probes. For this, follow general prehybridization and hybridization procedures.
    1. Carry out prehybridization using ULTRAhyb-Oligo buffer for 30 min to one hour at 42 °C with gentle agitation.
      Note: Probe signal strength obtained for small RNAs may vary depending on the composition of the hybridization buffer used.
    2. End-label probes with 32P; a number of kits are commercially available but we used the Megaprime DNA labeling system and followed the manufacturer’s instructions. Hybridize the membrane with the prepared 32P-end-labelled oligonucleotide probes. The probes must be labeled at high specific activity (≥ 108 cpm/μg template).
    3. Hybridize the membrane overnight (14-16 h) at 38 to 42 °C with gentle agitation.
    4. Posthybridization, the membrane is washed twice with a low stringency buffer solution (2x SSC, 0.5% SDS) for 10 min each, and once using a high stringency buffer solution (0.1x SSC, 0.1% SDS) for 5 min at 42 °C. If necessary, washing time can be increased under more stringent conditions (e.g., when high background levels are seen).
    5. Expose the membrane to X-ray film at -80 °C for signal visualization. Adjust the exposure time depending on signal intensity.
      Note: It is extremely important that all steps involving radioactive material are followed under appropriate safety guidelines.

Recipes

  1. DEPC treated water
    Dissolve 1 ml of DEPC in 1 L of distilled water with continuous stirring and autoclave
  2. 10x TBE (1 liter)
    108 g Tris Base
    55 g Boric acid
    40 ml EDTA (0.5 M, pH 8.0)
    Bring volume to 1 liter with DEPC-treated water, mix by stirring and autoclave
  3. 10% APS
    Mix 1 g APS in 10 ml of RNase free water and filter sterilize
  4. 15% polyacrylamide gel
    21 g urea
    5 ml 10x TBE
    18.8 ml 40% Acrylamide/Bis-acrylamide (19:1)
    Bring volume to 50 ml with RNase free water and vortex well until urea is dissolved
    When ready to pour gel add by swirling briefly
    250 μl 10% Ammonium persulfate
    50 μl TEMED
    Note: TEMED should be added last. Mix the solution well, immediately pour gel smoothly without creating any air bubbles and let it polymerize. This recipe is sufficient to fill four mini gel cassettes. Amount may be adjusted depending on the application. Gel can be stored in 4 °C degree fridge after tightly wrapping in saran wrap with comb still inserted. Do not freeze gels.
  5. 2x loading buffer (50 ml)
    47.5 ml 95% Formamide
    2 ml EDTA (20 mM, pH 8.0)
    0.025 g Bromophenol blue
    0.025 g Xylene cyanol

Acknowledgments

This protocol was adapted from the protocol described by Pall and Hamilton (2008). We are grateful for discussions with and technical advice from Mrs. Melanie Walker and Dr. Hélène Sanfaҫon.

References

  1. Pall, G. S. and Hamilton, A. J. (2008). Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6): 1077-1084.
  2. Panwar, V., McCallum, B. and Bakkeren, G. (2013a). Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus. Plant Mol Biol 81(6): 595-608.
  3. Panwar, V., McCallum, B. and Bakkeren, G. (2013b). Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to the suppression of rust diseases on wheat. Plant J 73: 521-532.

材料和试剂

  1. 病毒感染的植物组织
  2. TRIzol试剂(Invitrogen)
  3. 氯仿(Sigma-Aldrich)
  4. 异丙醇(Sigma-Aldrich)
  5. 乙醇(EtOH)
  6. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich)
  7. Hybond NX Neutral Membrane(Amersham biosciences,目录号:RPN203T)
  8. 40%丙烯酰胺/N'N'-双 - 亚甲基 - 丙烯酰胺(19:1)(Life Technologies,Ambion )
  9. 四甲基乙二胺(EDTA)(Sigma-Aldrich)
  10. 脲(Sigma-Aldrich)
  11. Hyperfilm TM MP(Amersham biosciences,目录号:28-9068-45)
  12. 溴化乙锭(Sigma-Aldrich)
  13. 乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)(Sigma-Aldrich,目录号:39391)
  14. 过硫酸铵(APS)
  15. 四甲基乙二胺(TEMED)(Sigma-Aldrich)
  16. Tris碱(AMRESCO)
  17. 硼酸(Fisher Scientific)
  18. 甲酰胺(Sigma-Aldrich)
  19. 溴酚蓝(Sigma-Aldrich)
  20. 二甲苯蓝(Sigma-Aldrich)
  21. 3 MM Whatman过滤纸
  22. 1-甲基咪唑(Sigma-Aldrich)
  23. 盐酸(HCl)
  24. 十二烷基硫酸钠(SDS)(Fisher Scientific)
  25. 20x盐水柠檬酸钠(SSC)缓冲液(Sigma-Aldrich)
  26. ULTRAhyb-Oligo缓冲液(Life Technologies,Ambion ,目录号:AM8669)
  27. Megaprime DNA标记系统(通用电气公司,型号:RPN1604)
  28. 液氮
  29. 无RNase水
  30. 小凝胶(规则蛋白凝胶的尺寸为1.5mm厚)(Bio-Rad,Mini-Protean Cell)
  31. 中性Hybond硝酸纤维素膜
  32. RNase ZAP(Life Technologies,目录号:AM9784)
  33. DEPC处理的水(见配方)
  34. 10x Tris-硼酸盐-EDTA(TBE)缓冲液(参见配方)
  35. 10%APS(参见配方)
  36. 15%聚丙烯酰胺凝胶(见配方)
  37. 2x加载缓冲液(见配方)

设备

  1. 杵和臼
  2. 半干电吸印机(Bio-Rad)
  3. Protean II垂直凝胶系统(Bio-Rad)
  4. Nanodrop分光光度计
  5. 离心机
  6. 杂交炉
  7. UV透射仪
  8. 微量离心管
  9. 电泳仪
  10. X光片

程序

  1. RNA分离
    1. 使用研钵和研杵在液氮中研磨50-100mg病毒感染的植物组织。 立即加入1ml TRIzol试剂,继续研磨直到形成均匀的糊状物。 吸取均质材料到无菌微量离心管中,在25-30°C孵育5分钟,以允许核蛋白复合物完全解离。
    2. 加入200μl氯仿/ml TRIzol试剂,用于样品组织匀浆。 用手转动微量离心管数次,摇动样品,在20-30℃下孵育2-3分钟。
    3. 在室温(20-25℃)下将样品以不超过12,000×g离心15分钟。
    4. 离心后,混合物在低级红色酚 - 氯仿相,中间相和无色上层水相中分离。
    5. 在不干扰中间相的情况下,小心地移出水相并将其转移到新的无菌微量离心管中。
    6. 通过每ml所用的TRIzol试剂加入0.5ml异丙醇从水相中沉淀RNA
    7. 孵育样品在20-30°C 10分钟。通过在2-8℃下以不超过12,000×g离心样品10分钟来回收总RNA。 RNA将在微量离心管的底部和侧面形成凝胶状沉淀
    8. 不干扰沉淀去除上清液。通过在管的侧面缓慢加入1ml的75%乙醇洗涤RNA沉淀
    9. 通过涡旋混合样品,并在2-8℃下以不超过7,500×g离心5分钟。
    10. 小心弃去上清液,不影响沉淀。真空或空气干燥沉淀10-15分钟,并重悬在50μl无RNase的水(市售或DEPC处理的无菌水)。 体积是可调节的,但是较高浓度是优选的,因为这将允许较低体积加载到凝胶上。不要过度干燥颗粒,否则将难以溶解。通过使用分光光度计(例如,Nanodrop)测量260nm处的吸光度来定量RNA的浓度,并储存在-20℃直至准备使用。 RNA将在这些条件下稳定数月
  2. 15%聚丙烯酰胺凝胶电泳(PAGE)
    小凝胶(规则蛋白凝胶的尺寸为1.5mm厚)良好以检测小RNA的表达。然而,为了分析单个小RNA的长度变体(例如20nt,21nt,22nt),需要运行更长的凝胶。在组装电泳系统之前,使用RNase ZAP彻底清洁所有设备以进行任何RNase污染。通过使用无菌溶液和不含RNase的塑料制品避免整个程序中的核酸酶污染
    1. 根据制造商的说明和handcast 15%聚丙烯酰胺凝胶装配凝胶铸造室。让凝胶在室温(20-25℃)下聚合至少30分钟至1小时。根据制造商的建议设置电泳模块。使用注射器用流动缓冲液(0.5×TBE)彻底冲洗孔以除去任何痕量的尿素,并在装载样品之前用跟踪染料预运行凝胶约15分钟。
    2. 在预运行凝胶时,制备RNA样品。理想情况下,为了良好的siRNA分辨率,建议加载35-40μg总RNA(基于RNA的纯度和浓度)。因此,使用DEPC处理的水调节待装载的每个样品(根据最小浓缩的样品)的体积。向样品中加入等体积的2x负载染料。通过轻轻敲击轻轻混匀,然后短暂离心
    3. 盖上管并通过在90-100℃加热15分钟使RNA样品变性。简单地旋转样品,然后在冰上快速冷却5分钟
    4. 加载样品和siRNA大小标记,并在200V运行凝胶约一小时。
      注意:凝胶的电压和持续时间取决于所使用的电源和凝胶电泳系统的类型。当溴酚蓝染料迁移到凝胶的末端时停止运行。不要超过。

  3. 半干电印迹到尼龙膜上的siRNA
    1. 小心拆卸电泳仪器,不要破坏凝胶。切割含有跟踪染料(二甲苯蓝和溴酚蓝)条带的凝胶的下部。在15%聚丙烯酰胺凝胶上,溴酚蓝染料前面运行接近10nt的长RNA。包含tRNA和5S RNA的凝胶的上部用溴化乙锭溶液(0.25μg/ml溴化乙锭在0.5x TBE缓冲液中)染色10-20分钟。使用360 nm紫外透射仪检查凝胶中RNA的完整性和等量加载。 tRNA条带的清晰度和清晰度是RNA质量和完整性的良好指标(图1)

      图1.显示23个核苷酸的小干扰RNA分子的Northern印迹(上图)。在RNA转移前,溴化乙锭染色的rRNA在凝胶中作为加样对照(下图)。

    2. 为了制备转移夹心,将3MM Whatman滤纸(六片)和中性Hybond硝酸纤维素膜切成凝胶大小。将膜和Whatman纸浸泡在0.5x TBE缓冲液中,直到它们完全润湿。膜的完全润湿对于确保核酸的适当结合是重要的。组装三明治如下。
    3. 将三块Whatman纸放在电印机的阳极板上。确保滚出可能抑制转印的气泡。
    4. 将膜转移到滤纸的顶部并且滚出在膜和滤纸之间形成的任何气泡。电泳后,将凝胶在转移缓冲液(0.5x TBE)中平衡5至10分钟。平衡有助于去除电泳缓冲盐和去污剂。小心地将平衡的凝胶放在膜上,使叠层尽可能完美
    5. 将在运行缓冲液中饱和的剩余三块Whatman纸放在纸叠上,并滚出任何残留的空气
    6. 用转移缓冲液(0.5×TBE)稍微润湿该叠层,并将阳极板放在顶部,而不干扰凝胶 - 硝化纤维素叠层并牢固地固定。
      注意:重要的是在设置转印时,排除滤纸和隔膜中夹带的多余缓冲液和气泡。
    7. 设置电源并在10 V和200 mAmp(恒定电流设置)下运行传输单元30分钟。小心地拆卸传输单元,取出滤纸和凝胶在尼龙膜的顶部。在RNA转移侧用铅笔标记膜上的凝胶槽的取向。

  4. 化学交联
    RNA与膜的交联经常提高Northern印迹的灵敏度。可使用常规方法例如暴露于标准剂量的UV(120mJ/cm 2)或通过在烘箱中烘烤(80℃下30分钟)将RNA固定到膜上。这两种方法都可以成功使用,但是使用基于EDC的小RNA对膜的交联通过杂交大大提高了小RNA的信号分辨率(Pall和Hamilton,2008)。
    1. 在即将使用前,制备0.16M EDC溶液在0.13M 1-甲基咪唑中的溶液,并用HCl调节溶液的pH至8。
    2. 切割一张3MM的Whatman滤纸,比膜的尺寸略大,并用EDC溶液使其饱和。
    3. 将膜置于EDC饱和的3MM纸上,RNA的面向上转移到侧面。 推出夹在膜和滤纸之间的任何气泡
    4. 盖上托盘,用膜包裹膜和滤纸,并在60℃下孵育1至2小时。
    5. 取出膜,用无RNA酶的水冲洗,以除去任何残留的EDC溶液
    6. 可以在去除残留的EDC之后将膜干燥并储存在-20℃下,而不损害siRNA检测的灵敏度,或立即用于杂交。

  5. 杂交
    最后,将具有固定RNA的膜与特异性放射性标记的探针一起温育。为此,遵循一般的预杂交和杂交程序
    1. 使用ULTRAhyb-Oligo缓冲液在42℃下轻轻搅拌30分钟至1小时进行预杂交。
      注意:对于小RNA获得的探针信号强度可以根据所使用的杂交缓冲液的组成而变化。
    2. 带有 P的末端标记探针;许多试剂盒可商购获得,但我们使用Megaprime DNA标记系统并遵循制造商的说明书。将膜与制备的32 P端标记的寡核苷酸探针杂交。探针必须以高比活性(≥10 8 cpm /μg模板)标记。
    3. 杂交膜过夜(14-16小时),在38至42℃,轻轻搅拌。
    4. 杂交后,将膜用低严格性缓冲溶液(2×SSC,0.5%SDS)洗涤两次,每次10分钟,并在42℃下使用高严格性缓冲液(0.1×SSC,0.1%SDS)洗涤5分钟。如果需要,可以在更严格的条件下(例如,当观察到高背景水平时)增加洗涤时间。
    5. 将膜暴露于-80°C的X射线胶片,用于信号可视化。 根据信号强度调整曝光时间。
      注意:所有涉及放射性物质的步骤都必须遵循适当的安全准则,这一点非常重要。

食谱

  1. DEPC处理水
    在连续搅拌和高压釜下将1ml DEPC溶解在1L蒸馏水中
  2. 10x TBE(1升)
    108克Tris碱
    55克硼酸
    40ml EDTA(0.5M,pH8.0) 用DEPC处理的水使体积达到1升,通过搅拌和高压釜混合
  3. 10%APS
    混合1克APS在10毫升无RNA酶的水和过滤灭菌
  4. 15%聚丙烯酰胺凝胶 21克尿素
    5 ml 10x TBE
    18.8ml 40%丙烯酰胺/双丙烯酰胺(19:1) 用无RNA酶的水将体积加至50ml,涡旋,直到尿素溶解 当准备好倾注凝胶时,通过短暂旋转
    250μl10%过硫酸铵
    50μlTEMED
    注意:TEMED应该最后添加。 混合溶液,立即倒入凝胶,不产生任何气泡,让它聚合。 这个配方足以填充四个迷你凝胶盒。 量可以根据应用调整。 凝胶可以紧紧包裹在saran包装中,然后插入梳子储存在4℃冰箱中。 不要冻结凝胶。
  5. 2x加样缓冲液(50ml)
    47.5ml 95%甲酰胺 2ml EDTA(20mM,pH8.0) 0.025g溴酚蓝
    0.025克二甲苯蓝

致谢

该方案改编自Pall和Hamilton(2008)描述的方案。 我们非常感谢Melanie Walker女士和HélèneSanfaҫon博士的讨论和技术咨询。

参考文献

  1. Pall,G.S。和Hamilton,A.J。(2008)。 改进的Northern印迹方法,用于增强小RNA的检测。 Nat Protoc 3(6):1077-1084。
  2. Panwar,V.,McCallum,B。和Bakkeren,G。(2013a)。 小麦叶锈病真菌宿主诱导的基因沉默小麦叶锈病病原性基因 81 81(6):595-608。
  3. Panwar,V.,McCallum,B。和Bakkeren,G。(2013b)。 通过 致病性基因的内源性沉默

    植物表达序列导致抑制小麦锈病。 73 73:521-532。
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How to cite this protocol: Panwar, V. and Bakkeren, G. (2013). A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants. Bio-protocol 3(20): e940. DOI: 10.21769/BioProtoc.940; Full Text



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