Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue

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Molecular Plant Microbe Interactions
Sep 2014



This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.

Keywords: In situ RT-PCR (原位RT-PCR检测), Gene expression (基因表达的影响), Syncytium (合胞体), Nematode feeding site (线虫喂养现场), Heterodera schachtii (大豆胞囊线虫schachtii)

Materials and Reagents

  1. Arabidopsis roots
    Note: Non-infected root fragments or root fragments containing feeding sites of H. schachtii (syncytia) or M. incognita (giant-cells embedded within galls).
  2. Ethanol
  3. Formaldehyde
  4. Diethylpyrocarbonate-treated water (DEPC-H2O)
  5. Low melting agarose
  6. Petri dishes ( 50 mm)
  7. 12/24-well plates
  8. Parafilm
  9. Superglue
  10. RNase Away (Thermo Fisher Scientific-Molecular BioProducts, catalog number: 7005 )
  11. DNase I, RNase-free, HC (50 U/µl) (50 U/ul, 1,000 U) ((Thermo Fisher Scientic, Fermentas, catalog number: EN0523 )
  12. RiboLock RNase Inhibitor (40 U/µl) (RiboLock: 40 U/µl) (Thermo Fisher Scientic, Fermentas, catalog number: EO0381 )
  13. Ethylenediaminetetraacetic acid (EDTA)
  14. SuperScriptTM III Reverse Transcriptase Kit (Life Technologies, InvitrogenTM, catalog number: 18080-093 )
  15. Deoxynucleotides (dNTPs)
  16. Bovine serum albumin (BSA)
  17. DIG (Digoxigenin-11dUTP-alkali stable; 25 nmol/25 µl) (Roche Diagnostics, catalog number: 11093088910 )
  18. BioThermTM Taq DNA Polymerase (GeneCraft, catalog number: GC-002-0100 )
  19. Anti-Digoxigenin-AP, Fab fragments (150 U/200 µl) (Roche Diagnostics, catalog number: 11093274910 )
  20. BCIP®/NBT Liquid Substrate System (Sigma-Aldrich, catalog number: B1911-100 ML )
  21. Fixation solution (see Recipes)
  22. 10x phosphate buffered saline (PBS) (see Recipes)
  23. 20x SSC (see Recipes)
  24. 10x PCR buffer (see Recipes)
  25. 10x washing buffer (see Recipes)


  1. Vibratome (Leica, model: VT 100 )
  2. Forceps
  3. Heating plate
  4. Laminar flow
  5. PCR cycler
  6. Stereo microscope


Precautions: RNA is very sensitive and easily degraded by RNases! Therefore always wear gloves, use RNase-free water as well as glass- and plasticware. Recommended is the use of commercially available reagents for eliminating RNase and DNA contamination from the surface of glassware or plasticware e.g. RNase away!

  1. Fixation
    1. Cut segments of Arabidopsis roots containing feeding sites of H. schachtii (syncytia) or M. incognita (giant-cells embedded within galls) and corresponding non-infected root fragments.
    2. Immediately put the root pieces into a microcentrifuge tube containing freshly prepared and pre-chilled fixation solution and vacuum infiltrate it on ice for 15-20 min to ensure the proper penetration of the fixative. Incubate samples 42 h or longer at 4 °C on a shaker with gentle shaking.

  2. Embedding
    1. Subsequently wash fixed samples 3 x 10 min in 63% ethanol in PBS and 1 x 10 min in PBS.
    2. Embed the tissue in low melting agarose (5% w/v in PBS) in small petri dishes ( 50 mm) placed on a heating plate (35-40 °C) in the laminar flow. Grab the root fragments with the forceps, swab them carefully with a paper towel to remove the excess of PBS and dip them into warm agarose parallel to the bottom of the Petri dish or 12/24-well plate and approx. at the half depth of the agarose. Approx. 10 root segments/dish or 1-2 root segments/12/24-well plate (Figure 1A).
    3. Cool down the agarose in the laminar flow.
    4. Petri dishes sealed with Parafilm can be stored in the fridge (4 °C) for several weeks.

  3. Preparation of cross sections with the vibratome and DNase treatment
    1. Cut out small agarose blocks (approx. 8 mm high x 5 mm wide) containing the syncytia or non-infected root segments with a scalpel. Trim the agarose block under the microscope (Figure 1B) and glue it on the round vibratome plate with a drop of common superglue. To produce cross sections the root segments within the agarose block have to be placed perpendicular to the bottom of the vibratome plate (Figure 1B-C).

      Figure 1. Placement of the root fragments within a well of the 12-well plate (A; view from above), trimmed agarose block with a root fragment in the perpendicular position (B; view from the side) and agarose block glued onto the vibratome plate (C)

    2. Cut 20-25 µm thick tissue sections in the vibratome tray filled with DEPC-H2O.
      Using forceps (in case the tissue section remains in the agarose section) or a pipette (in case the tissue section disconnects from the agarose section) collect the sections in a watchglass or similar container filled with DEPC-H2O supplemented with RNase inhibitor.
    3. With a pipette remove the excess of DEPC-H2O (as much as possible). To prevent non-specific DNA signals digest the DNA within the tissue by adding 2 µl RNase-free DNAse and 1 µl RNase Inhibitor to the remaining DEPC-H2O covering the cross-sections. Subsequently incubate the samples overnight at 37 °C in a watchglass in a Petri dish sealed with parafilm.

  4. Washing
    1. Add several drops of 0.5 M EDTA and incubate the cross-sections at 65 °C for 10 min to inactivate the DNase.
      Subsequently wash sections as follows:
      2 times with 2x SSC buffer
      1 time with 1x SSC buffer
      1 time with 0.5x SSC buffer
      2 times with ddH2O
      Each step should be performed with 1 ml of buffer or ddH2O in a sealed Petri dish at 37 °C for 10 min.

  5. Reverse transcription
    1. Transfer several sections into a PCR tube using a pipette (in ca. 11.5 µl ddH2O).
    2. Add 0.5 µl 3’ primer and 1.0 µl dNTPs and start the first step of the RT reaction: 65 °C for 5 min.
    3. Cool down the samples, incubate them at least 1 min on ice and add 4 µl of the 5x First-Strand Buffer, 1 µl 0.1 M DTT, 1 µl RNase Inhibitor and 1 µl SuperScriptTM Reverse Transcriptase. Total volume 20 µl.
    4. Run the following program in a PCR cycler:
      25 °C - 5 min
      55 °C - 1 h
      75 °C - 5 min (inactivation of DNase)

  6. PCR
    1. Prepare a dNTPs mix containing 10 mM dGTP, dATP and dCTP. Add to the RT-mix as follows:
      5.0 µl - 10x PCR buffer
      1.0 µl - 3’ primer (final concentration 0.5 µM)
      1.0 µl - 5’ primer (final concentration 0.5 µM)
      1.0 µl - dNTPs mix
      2.36 µl - 2 mM dTTP
      0.5 µl - DIG
      0.25 µl - BioThermTM Taq DNA Polymerase
      18.89 µl - ddH2O
      Total volume 50 µl (30 µl of PCR-mix and 20 µl of RT-mix).
      In case of the negative control the polymerase will be omitted.

  7. Washing and detection
    The following steps are carried out under the stereo microscope at room temperature (from step G5 in dark room with a weak source of light e.g. small table lamp). Short centrifugation could be necessary between each washing step to collect the sections at the bottom of the PCR tube. This procedure might prevent the accidental removal of tissue sections during several washing rounds. You might also check the discarded liquids for unintended removed sections after each washing step.
    1. Wash the samples twice in 1x PBS buffer for 5 min.
    2. To block unspecific binding sites incubate the sections for 30 min in freshly prepared blocking solution containing 0.1% w/v BSA in 1x PBS.
    3. Remove the blocking solution and add 50 µl Anti-DIG-AP, Fab fragments diluted 1:500 in blocking solution. Incubate 1 h at RT.
    4. Remove the blocking solution containing Anti-DIG-AP, Fab fragments and wash the sections 2 x 15 min with 10x washing buffer.
    5. Using a pipette place a drop of the washing buffer containing section(s) on a microscopic slide and replace the washing buffer with 50 µl of the NBT/BCIP solution (1:50 in washing buffer). Keep the slide in darkness (use e.g. lid from chemical bottle as a cover) since the NBT/BCIP is light sensitive!
    6. From time to time observe the progress of the staining reaction under the microscope, it appears usually within first 2-3 min (keep the light exposure as short as possible).
    7. If the violet staining is clearly visible stop the reaction with ddH2O and photograph your sections. Figure 2 shows an example of an in situ RT-PCR analysis made for two β-1, 4- endoglucanases, AtCel1 and KOR3, which are specifically up-regulated in syncytia induced by H. schachtii in Arabidopsis roots.

      Figure 2. In situ RT-PCR analysis of AtCel2 and KOR3 on sections of syncytia induced by Heterodera schachtii at 10 days after inoculation (dai). (a) AtCel2 transcripts accumulate within syncytium. (b) Control reaction for (a) performed without polymerase. Staining is not detected. (c) Control reaction for (a) on a root section above the syncytium. Transcripts of AtCel2 were not detected. (d) Strong staining associated with transcripts of KOR3 is visible in the syncytium. (e) Control reaction to (d) performed without polymerase; no staining is visible. (f) Control reaction for (d) on a root section above the syncytium. No transcripts of KOR3 are visible. (a), (b), (d) and (e) scale bar = 50 µm; (c) and (f) scale bar = 20 µm. From Wieczorek et al. (2008), with permission.


  1. RNA is easily degraded by RNAses and several different feeding sites/roots should be used to enhance the success rate of this method. However, if the feeding site/root tissue that is used contains intact RNA the reproducibility of this protocol is high and the variability low.
  2. Start the detection with the positive reaction and subsequently use exactly the same exposure time for the controls.
  3. The coloration of gene transcript-specific staining may vary from light violet to dark violet (almost black). The unspecific staining in the control sections (without Taq Polymerase) may vary from light brown, reddish to very faint/almost transparent.


  1. Fixation solution
    63% ethanol
    5% formaldehyde in PBS (containing DEPC-ddH2O, pH 7.2)
  2. 10x phosphate buffered saline (PBS)
    10 mM Na2HPO4
    130 mM NaCl in DEPC-ddH2O
    pH 7.5
  3. 20x SSC
    3 M NaCl
    300 mM C6H5O7.2H2O.3Na (sodium citrate dihydrate) in DEPC-ddH2O
  4. 10x PCR buffer
    160 mM (NH4)2SO4
    670 mM Tris-HCl (pH 8.8) (at 25 °C)
    15 mM MgCl2
    0.1% Tween 20
    5. 10x washing buffer
    0.1 M Tris-HCl
    0.15 M NaCl
    pH 9.5


This protocol is a combination of methods previously published by Koltai and Bird (2000) and Urbanczyk-Wlochniak et al. (2002) and has been additionally modified. This work was supported by grant QLK-CT-1999-01501 (‘NONEMA’) from the European Union within the 5th Framework and FWF grants P16296-B06, P16897-B06 and P21067-B12.


  1. Hofmann, J., Wieczorek, K., Blochl, A. and Grundler, F. M. (2007). Sucrose supply to nematode-induced syncytia depends on the apoplasmic and symplasmic pathways. J Exp Bot 58(7): 1591-1601.
  2. Koltai, H. and Bird, D. M. (2000). High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections. Plant Physiol 123(4): 1203-1212.
  3. Siddique, S., Endres, S., Atkins, J. M., Szakasits, D., Wieczorek, K., Hofmann, J., Blaukopf, C., Urwin, P. E., Tenhaken, R., Grundler, F. M., Kreil, D. P. and Bohlmann, H. (2009). Myo-inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in Arabidopsis roots. New Phytol 184(2): 457-472.
  4. Urbanczyk-Wochniak, E., Filipecki, M. and Przybecki, Z. (2002). A useful protocol for in situ RT-PCR on plant tissues. Cell Mol Biol Lett 7(1): 7-18.
  5. Wieczorek, K., Golecki, B., Gerdes, L., Heinen, P., Szakasits, D., Durachko, D. M., Cosgrove, D. J., Kreil, D. P., Puzio, P. S., Bohlmann, H. and Grundler, F. M. (2006). Expansins are involved in the formation of nematode-induced syncytia in roots of Arabidopsis thaliana. Plant J 48(1): 98-112.
  6. Wieczorek, K., Hofmann, J., Blochl, A., Szakasits, D., Bohlmann, H. and Grundler, F. M. (2008). Arabidopsis endo-1,4-beta-glucanases are involved in the formation of root syncytia induced by Heterodera schachtii. Plant J 53(2): 336-351.


该方案描述了原位 RT-PCR的有效方法,其被开发用于直接在由胞囊线虫Heterodera schachtii诱导的线虫摄食位点的薄横截面中定位特异性基因表达。 在拟南芥根中( H。schachtii )或根结线虫 Meloidogyne incognita 使用与AP(碱性磷酸酶)和硝基蓝四唑/5-溴-4-氯-3'-吲哚磷酸盐基检测偶联的DIG(地高辛-11dUTP)标记。 该方法适用于任何其他拟南芥根组织。

关键字:原位RT-PCR检测, 基因表达的影响, 合胞体, 线虫喂养现场, 大豆胞囊线虫schachtii


  1. 拟南芥根

    注意:包含H. schachtii(合胞体)或南方根结线虫
  2. 乙醇
  3. 甲醛
  4. 焦碳酸二乙酯处理水(DEPC-H 2 O)
  5. 低熔点琼脂糖
  6. 培养皿( 50 mm)
  7. 12/24孔板
  8. parafilm
  9. 超能量
  10. RNase Away(Thermo Fisher Scientific-Molecular BioProducts,目录号:7005)
  11. DNase I,无RNA酶,HC(50U /μl)(50U/ul,1,000U)(Thermo Fisher Scientific,Fermentas,目录号:EN0523)
  12. RiboLock核糖核酸酶抑制剂(40U /μl)(RiboLock:40U /μl)(Thermo Fisher Scientic,Fermentas,目录号:EO0381)
  13. 乙二胺四乙酸(EDTA)
  14. SuperScript TM反转录酶试剂盒(Life Technologies,Invitrogen TM ,目录号:18080-093)
  15. 脱氧核苷酸(dNTP)
  16. 牛血清白蛋白(BSA)
  17. DIG(洋地黄毒苷-11dUTP-碱稳定; 25nmol /25μl)(Roche Diagnostics,目录号:11093088910)
  18. BioTherm Taq DNA聚合酶(GeneCraft,目录号:GC-002-0100)
  19. 抗洋地黄毒苷-AP,Fab片段(150U /200μl)(Roche Diagnostics,目录号:11093274910)
  20. /PBS/NBT液体基质系统(Sigma-Aldrich,目录号:B1911-100ML)中。
  21. 固定解决方案(参见配方)
  22. 10x磷酸盐缓冲盐水(PBS)(见Recipes)
  23. 20x SSC(请参阅配方)
  24. 10x PCR缓冲液(参见配方)
  25. 10x洗涤缓冲液(见配方)


  1. Vibratome(Leica,型号:VT 100)
  2. 镊子
  3. 加热板
  4. 层流
  5. PCR循环仪
  6. 立体显微镜


注意事项:RNA非常敏感,容易被RNA酶降解! 因此,始终戴手套,使用无RNase的水以及玻璃和塑料制品。 推荐使用市售试剂从玻璃器皿或塑料制品表面消除RNase和DNA污染。 RNase离开!

  1. 固定
    1. 切割含有H的饲养位点的拟南芥根部。 schachtii (syncytia)或 M。 incognita (嵌入痣中的巨细胞)   和相应的未感染根段
    2. 立即放 将根块放入含有新鲜制备的微量离心管中 和预冷的固定溶液,并在冰上真空渗透 15-20分钟以确保固定剂的适当渗透。 孵化 样品在4℃下在摇动器上轻轻摇动42小时或更长时间

  2. 嵌入
    1. 随后在63%乙醇的PBS溶液中洗涤固定的样品3×10分钟,在PBS中洗涤1×10分钟
    2. 将组织嵌入低熔点琼脂糖(5%w/v的PBS)的小 置于加热板(35-40℃)上的培养皿( 50mm) 在里面 层流。 用镊子抓住根碎片,拭拭他们 小心地用纸巾除去过量的PBS并将其浸泡 进入平行于培养皿或12/24-孔底部的温热琼脂糖中   板和约。 在琼脂糖的一半深度。 大约。 10根 段/碟或1-2根段/12/24孔板(图1A)
    3. 在层流中冷却琼脂糖。
    4. 用石蜡膜密封的陪替氏培养皿可以在冰箱(4℃)中储存数周。

  3. 用vibratome和DNase处理的横截面的制备
    1. 切出小琼脂糖块(约8毫米高×5毫米宽)含 合胞体或未感染的根段与手术刀。 修剪 琼脂糖块在显微镜下(图1B)并胶合在圆上 vibratome板与一滴共同的superglue。 生成交叉 必须放置琼脂糖块中的根段 垂直于振动板的底部(图1B-C)

      图1.将根片段置于12孔的孔内 板(A;从上面看),修剪的琼脂糖块与根片段 在垂直位置(B;从侧面看)和琼脂糖块 胶合到振动板(C)上

    2. 在填充有DEPC-H 2 O的vibratome托盘中切割20-25μm厚的组织切片。
      使用镊子(万一组织切片保留在琼脂糖 部分)或移液管(在组织切片从 琼脂糖切片)在表玻璃或类似物中收集切片 容器,其填充有补充有RNA酶抑制剂的DEPC-H 2 O
    3. 用移液管除去过量的DEPC-H 2 O(尽可能多的)。 至 防止非特异性DNA信号消化组织内的DNA 加入2微升无RNA酶的DNAse和1微升RNA酶抑制剂剩余 覆盖横截面的DEPC-H 2 O 2。 随后孵育样品 在用石蜡膜密封的培养皿中的表玻璃中于37℃过夜。

  4. 洗涤
    1. 加入几滴0.5 M EDTA,孵育横截面在65°C 10分钟灭活DNase。
      用2x SSC缓冲液洗涤2次
      1次使用1x SSC缓冲区
      1次用0.5x SSC缓冲液
      用ddH <2> O 2次/2次 每个步骤应当在密封的培养皿中在37℃下用1ml缓冲液或ddH 2 O进行10分钟。

  5. 反转录
    1. 使用移液管(在约11.5μlddH 2 O)将几个部分转移到PCR管中。
    2. 加入0.5μl3'引物和1.0μldNTP,并开始RT反应的第一步:65℃5分钟。
    3. 冷却样品,在冰上孵育至少1分钟,加入4   μl5x第一链缓冲液,1μl0.1M DTT,1μlRNA酶抑制剂 和1μlSuperScript TM Reverse Transcriptase。 总体积20μl。
    4. 在PCR循环仪中运行以下程序:
      25°C - 5分钟
      55°C - 1小时
      75°C - 5分钟(DNase失活)

  6. PCR
    1. 制备含有10mM dGTP,dATP和dCTP的dNTPs混合物。 添加到RT-mix如下:
      5.0μl - 10x PCR缓冲液
      1.0μl-5'引物(终浓度0.5μM) 1.0μl - dNTPs mix
      2.36μl - 2 mM dTTP
      0.5μl - DIG
      0.25μl-BioTherm TM Taq DNA聚合酶
      18.89μl-ddH 2 O
      总体积50μl(30μlPCR混合物和20μlRT-混合物) 在阴性对照的情况下,聚合酶将被省略

  7. 清洗和检测
    以下步骤在室温下在立体显微镜下进行(来自具有弱光源例如小台灯的暗室中的步骤G5)。 在每个洗涤步骤之间可能需要短离心以收集PCR管底部的切片。 该程序可以防止在几个洗涤循环期间意外移除组织切片。 您还可以在每个清洗步骤后检查废弃的液体是否意外取出。
    1. 用1×PBS缓冲液洗涤样品两次,每次5分钟
    2. 阻止 非特异性结合位点在新鲜孵育切片30分钟 制备的含有0.1%w/v BSA的1x PBS溶液的封闭溶液
    3. 取出封闭溶液,加入在封闭溶液中1:500稀释的50μl抗DIG-AP , Fab片段。 在室温下孵育1小时。
    4. 取出含有抗DIG-AP , Fab片段的封闭溶液,并用10x洗涤缓冲液洗2次15分钟。
    5. 使用吸管放置一滴含有洗涤缓冲液 在微量载玻片上切片并用50稀释洗涤缓冲液   μlNBT/BCIP溶液(在洗涤缓冲液中1:50)。 保持幻灯片   黑暗(例如使用化学瓶盖作为盖子) NBT/BCIP是光敏感的!
    6. 经常观察 进展的显微镜下的染色反应,出现 通常在2-3分钟内保持曝光短 可能)。
    7. 如果紫色染色清晰可见,停止 与ddH 2 O 2反应并拍摄你的部分。 图2显示了a 用于两个β-1,4-半乳糖苷酶的原位 RT-PCR分析的实例 内切葡聚糖酶,AtCel1 和 KOR3 ,其是特异性上调的

      图2. 对合胞体诱导的部分的 AtCel2 和 KOR3 的原位 RT-PCR分析 (a) 在AtCel2 (b)(a) 无聚合酶。未检测到污点。 (c)控制 在合胞体上方的根部上的(a)的反应。未检测到 AtCel2 的誊本。 (d)强染色 在合胞体中可以看到 KOR3 的转录物。 (e)控制反应  (d)不用聚合酶进行;没有可见的染色。 (f)控制 (d)在合胞体上方根部的反应。没有成绩单 的 KOR3 可见。 (a),(b),(d)和(e)比例尺=50μm; (c)和 (f)比例尺=20μm。来自Wieczorek等人(2008),经许可。


  1. RNA容易被RNA酶降解,并且应当使用几个不同的进食位点/根以增强该方法的成功率。 然而,如果使用的饲养部位/根组织包含完整的RNA,则该方案的可重复性高且可变性低。
  2. 用阳性反应开始检测,随后对于对照使用完全相同的曝光时间
  3. 基因转录物特异性染色的着色可以从浅紫色到深紫色(几乎黑色)不同。 对照切片(无Taq聚合酶)中的非特异性染色可以从浅棕色,微红至非常微弱/几乎透明。


  1. 固定溶液
    PBS中的5%甲醛(含有DEPC-ddH 2 O,pH7.2)
  2. 10x磷酸盐缓冲盐水(PBS)
    10mM Na 2 HPO 4
    130mM NaCl,在DEPC-ddH 2 O中 pH 7.5
  3. 20x SSC
    3 M NaCl
    300mM C 6 H 5 O 7,H 2 O 7,H 2 O 3,H 2 O 3, 。在DEPC-ddH 2 O 2中的3Na(柠檬酸钠二水合物)
  4. 10x PCR缓冲液
    160mM(NH 4)2 SO 4 4
    670mM Tris-HCl(pH8.8)(在25℃) 15mM MgCl 2·h/v 0.1%Tween 20
    0.1M Tris-HCl
    0.15 M NaCl
    pH 9.5


该方案是由Koltai和Bird(2000)和Urbanczyk-Wlochniak等人(2002)先前公布的方法的组合,并且已经被另外修饰。 这项工作得到了来自欧盟在第五框架内的授权QLK-CT-1999-01501('NONEMA')和FWF授予P16296-B06,P16897-B06和P21067-B12的支持。


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引用:Wieczorek, K. (2015). Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue . Bio-protocol 5(18): e1597. DOI: 10.21769/BioProtoc.1597.