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Total RNA Isolation after Laser-capture Microdissection of Human Cervical Squamous Epithelial Cells from Fresh Frozen Tissue
从鲜冻组织中激光捕获显微解剖人宫颈上皮细胞后分离总RNA   

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Abstract

As most tissue specimens contain a mixture of different cell types including epithelial cells, stromal cells and immune cells, selection of the cells of interest is of utmost importance for the accurate determination of gene/microRNA expression. Laser capture microdissection enables the researcher to obtain homogeneous ultrapure cell selections from heterogeneous starting material. The following protocol was optimized for the isolation of total RNA from cervical (premalignant) squamous epithelial cells from fresh frozen biopsy specimens.

Keywords: LCM(LCM), RNA(RNA), Fresh frozen(新鲜冷冻)

Materials and Reagents

  1. Fresh frozen tissue specimens stored in liquid nitrogen
  2. Tissue-Tek O.C.T. (Sakura Finetek Europe B.V., catalog number: 4583 )
  3. Mayers’ Haematoxylin
  4. Ethanol
  5. Xylene
  6. Liquid nitrogen or dry ice
  7. TRIzol (Life Technologies, catalog number: 15596-026 ) or a preferred alternative RNA isolation method
  8. Glycogen (F. Hoffmann-La Roche, catalog number: 10901393001 )
  9. Chloroform
  10. Isopropanol (Isopropyl alcohol)
  11. RNase-free water

Equipment

  1. 21-26 gauge needle
  2. PEN foil 2.0 UM covered slides (Leica, catalog number: 11505158 )
  3. 50 ml tubes with screw cap (Greiner Bio-one, catalog number:  210261 )
  4. Cryotome (rotary microtome in a frozen section environment)
  5. Leica Laser Microdissection system (AS LMD, model: LMD6500 or LMD7000 )
  6. Centrifuge
  7. -80 °C freezer for sample storage

Procedure

  1. Take all necessary precautions for RNA handling throughout the whole procedure.
    1. Wear gloves at all times.
    2. Only use RNase free equipment, plastics and reagents.
  2. Pretreat the PEN foil slides with UV light for 15 min to reduce static electricity.
    Note: We use a UV source of 50 Hz and 26 Watt.
  3. Cut 8-10 μm thick sections from the frozen tissue specimens (embedded in Tissue-Tek) and apply sections directly to the PEN foil covered side of the slide.
    Notes:
    1. More than one section can be applied to the same slide depending on the size of the specimen.
    2. Make sure that all sections are within the rectangle where the foil is not glued to the slide.
    3. Keep sections dry and cold at all times (store in a closed 50 ml tube at -80 °C and transport in liquid nitrogen or on dry ice).
    4. In addition a 4 μm section for standard H&E staining can be made to allow for demarcation of the tissue area/cells of interest by a pathologist or other expert.
  4. Slides containing tissue sections should be stored in a closed 50 ml tube and kept cold at all times (store at -80 °C and transport in liquid nitrogen or on dry ice).
    Note: The use of 50 ml tubes avoids condensation on the slide thereby keeping the tissue dry which reduces RNA degradation.
  5. Stain sections with Mayers’ Haematoxylin for 1 min at room temperature (RT).
    Notes:
    1. Before staining let slides come to RT while still in the closed 50 ml tube.
    2. Be careful not to lose the tissue section. Staining is best done by pipetting the haematoxylin directly on the slide.
  6. Carefully rinse slide in sterile (RNase free) water.
  7. Dehydrate tissue sections by subsequent rinsing for 1 min each in 50%, 70% and 100% ethanol (can be done in 50 ml tubes) and an 8 min incubation in Xylene (use a glass container).
  8. Store slides in a closed 50 ml tube at -80 °C or continue with the microdissection.
  9. Add a little (10-20 μl) TRIzol to the tube cap in which the microdissected tissue will fall.
    Notes:
    1. Let slides come to room temperature while in the 50 ml tube right before starting the microdissection procedure for that slide.
    2. If an alternative RNA isolation method is preferred, TRIzol should be substituted by the first reagent used in that method.
  10. Microdissect all areas/cells of interest from the slide using at a 10x magnification (see Figure 1) using the following settings:
    Intensity: 46
    Speed: 5 (or lower)
    Offset: 5
    Aperture diff: 6


    Figure 1. Example picture of a cervical tissue section before (upper panel) and after (lower panel) laser capture microdissection of the dysplastic epithelium. The microdissected tissue in the cap is shown in the left panel.

  11. Add another 80-90 μl of TRIzol (or alternative isolation reagent) to the 0.2 ml tube containing the microdissected tissue and transfer tissue and TRIzol/alternative isolation reagent to a 1.5 ml tube.
    Note: If preferred by the researcher an alternative RNA isolation method can be used. However, this protocol was optimized using TRIzol.
  12. Add TRIzol to the tube to obtain a total volume of 1 ml.
    Note: Store at -80 °C or continue with RNA isolation.
  13. Add 1 μl glycogen and mix well (vortex for 10 seconds).
  14. Disrupt the tissue/cells and shear the genomic DNA with 10 passes through a 21-26 gauge needle.
  15. Add 0.2 ml of chloroform and vortex for 30 sec.
  16. Spin down for 5 min at full speed (12,000 x g) at RT.
  17. Transfer the aqueous phase to a clean tube.
  18. Add 0.5 ml of isopropanol to the aqueous phase and mix well.
  19. Incubate samples for at least 1 h at -20 °C for optimal precipitation of the RNA.
  20. Spin down for 15 min at full speed (12,000 x g) at RT.
  21. Discard supernatant
    Note: Careful not to disturb the pellet containing your RNA.
  22. Wash pellet with 200 μl of 70% ethanol.
  23. Spin down at full speed for 10 min at RT.
  24. Discard supernatant.
  25. Repeat steps 20-22.
    Note: This time it is important to completely remove the supernatant.
  26. Airdry the pellet.
    Note: If all supernatant in removed in step 23 this should only take a few minutes.
  27. Resuspend the pellet in 10-30 μl RNase free water.
  28. Store at -80 °C.

Acknowledgments

This protocol was optimised with the help of Elza de Bruin (de Bruin et al., 2005) and Muriel Verkuijten. This work was supported by the Centre for Medical Systems Biology (CMSB) in the framework of the Netherlands Genomic Initiative, Royal Netherlands Academy of Arts and Sciences, the VUMC-CCA institute of the VU University Medical Center, Amsterdam, The Netherlands (grant number CCA20085-04) and the Dutch Cancer Society (KWF, grant number VU2010-4668).

References

  1. de Bruin, E. C., van de Pas, S., Lips, E. H., van Eijk, R., van der Zee, M. M., Lombaerts, M., van Wezel, T., Marijnen, C. A., van Krieken, J. H., Medema, J. P., van de Velde, C. J., Eilers, P. H. and Peltenburg, L. T. (2005). Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles. BMC Genomics 6: 142.
  2. Wilting, S. M., de Wilde, J., Meijer, C. J., Berkhof, J., Yi, Y., van Wieringen, W. N., Braakhuis, B. J., Meijer, G. A., Ylstra, B., Snijders, P. J. and Steenbergen, R. D. (2008). Integrated genomic and transcriptional profiling identifies chromosomal loci with altered gene expression in cervical cancer. Genes Chromosomes Cancer 47(10): 890-905.
  3. Wilting, S. M., Snijders, P. J., Verlaat, W., Jaspers, A., van de Wiel, M. A., van Wieringen, W. N., Meijer, G. A., Kenter, G. G., Yi, Y., le Sage, C., Agami, R., Meijer, C. J. and Steenbergen, R. D. (2013). Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis. Oncogene 32(1): 106-116.

简介

由于大多数组织标本含有不同细胞类型的混合物,包括上皮细胞,基质细胞和免疫细胞,所以选择感兴趣的细胞对于精确测定基因/微小RNA表达是至关重要的。 激光捕获显微切割使研究者能够从异质起始材料中获得均匀的超纯细胞选择。 以下方案针对从新鲜冷冻的活检标本的宫颈(恶化前)鳞状上皮细胞中分离总RNA进行了优化。

关键字:LCM, RNA, 新鲜冷冻

材料和试剂

  1. 储存在液氮中的新鲜冷冻组织标本
  2. Tissue-Tek O.C.T. (Sakura Finetek Europe B.V.,目录号:4583)
  3. Mayers'haematoxylin
  4. 乙醇
  5. 二甲苯
  6. 液氮或干冰
  7. TRIzol(Life Technologies,目录号:15596-026)或优选的替代性RNA分离方法
  8. 糖原(F.Hoffmann-La Roche,目录号:10901393001)
  9. 氯仿
  10. 异丙醇(异丙醇)
  11. 无RNase水

设备

  1. 21-26号针
  2. PEN箔2.0μm覆盖载玻片(Leica,目录号:11505158)
  3. 50ml带螺旋盖的管(Greiner Bio-one,目录号:210261)
  4. 冷冻切片机(在冷冻切片环境中的旋转切片机)
  5. 徕卡激光显微切割系统(AS LMD,型号:LMD6500或LMD7000)
  6. 离心机
  7. -80°C冰箱用于样品存储

程序

  1. 在整个程序中对RNA处理采取所有必要的预防措施。
    1. 始终戴上手套。
    2. 只使用无RNase设备,塑料和试剂
  2. 用紫外线预处理PEN箔片15分钟以减少静电。
    注意:我们使用50 Hz和26 W的紫外线源。
  3. 从冷冻的组织标本(嵌入Tissue-Tek)中切出8-10μm厚的切片,并将切片直接施加到载玻片的PEN箔覆盖侧。
    注意:
    1. 根据样本的大小,可以将多个部分应用于同一张幻灯片。
    2. 始终保持干燥和冷却(储存在密闭的50ml管中,在-80℃下,在液氮或干冰中运输)。
    3. 此外,可以制备用于标准H& E染色的4μm切片,以允许由病理学家或其他专家划分感兴趣的组织区域/细胞。
  4. 含有组织切片的玻片应储存在封闭的50ml管中并保持冷(在-80℃保存并在液氮或干冰中运输)。
    注意:使用50ml管避免在载玻片上冷凝,从而保持组织干燥,从而减少RNA降解。
  5. 在室温(RT)下用Mayers的苏木精染色1分钟 注意:
    1. 在染色之前,让载玻片进入RT,同时仍然在封闭的50ml管中。
    2. 小心不要丢失组织切片。 染色最好通过直接在载玻片上吸取苏木精来进行。
  6. 小心冲洗无菌(无RNA酶)水滑梯。
  7. 通过随后在50%,70%和100%乙醇中漂洗1分钟(可在50ml管中进行)和在二甲苯中培养8分钟(使用玻璃容器)来脱水组织切片。
  8. 将载玻片置于密闭的50ml管中,在-80°C或继续显微切割
  9. 添加一点(10-20微升)TRIzol到管帽,其中显微切割组织将下降。
    注意:
    1. 在开始显微切割术之前,让玻片在50毫升管中达到室温。
    2. 如果选择其他RNA分离方法,TRIzol应该替换为该方法中使用的第一个试剂。
  10. 使用以下设置,以10倍放大率(参见图1),从载玻片上显微解剖感兴趣的所有区域/细胞:
    强度:46
    速度:5(或更低)
    偏移:5
    孔径差:6


    图1.子宫颈组织切片的实例图片在发育不良的上皮的激光捕获显微切割后 e(上图)和之后(下图)。帽子中的显微切割组织显示在左侧面板中。

  11. 添加另一个80-90微升的TRIzol(或替代分离试剂)到含有显微切割的组织和转移组织和TRIzol /替代隔离试剂的0.2毫升管到1.5毫升管。 注意:如果研究者喜欢,可以使用另一种RNA分离方法。 但是,此协议已使用TRIzol优化。
  12. 向管中加入TRIzol,得到总体积为1ml 注意:储存于-80°C或继续RNA分离。
  13. 加入1μl糖原并混匀(涡旋10秒钟)
  14. 破坏组织/细胞,并剪切基因组DNA与10通过21-26规格的针
  15. 加入0.2ml氯仿并涡旋30秒
  16. 在室温下全速旋转5分钟(12,000×g )。
  17. 将水相转移到干净的管中
  18. 向水相中加入0.5ml异丙醇,充分混合
  19. 在-20°C孵育样品至少1小时,以获得最佳的RNA沉淀
  20. 在室温下全速(12,000
  21. 丢弃上清液
    注意:小心不要打扰含有RNA的沉淀。
  22. 用200μl70%乙醇洗涤沉淀。
  23. 在室温下全速旋转10分钟。
  24. 弃去上清液。
  25. 重复步骤20-22。
    注意:这一次,彻底清除上清液很重要。
  26. 对球团造粒。
    注意:如果在步骤23中移除的所有上清液,这只需要几分钟。
  27. 用10-30μl无RNA酶的水重悬沉淀
  28. 储存于-80°C。

致谢

该方案在Elza de Bruin(de Bruin等人,2005年)和Muriel Verkuijten的帮助下进行了优化。这项工作得到了荷兰基因组倡议,荷兰皇家艺术和科学学院,VUMC-CCA研究所的VU大学医学中心,荷兰阿姆斯特丹(授权号码)框架下的医疗系统生物学中心(CMSB) CCA20085-04)和荷兰癌症协会(KWF,授权号VU2010-4668)。

参考文献

  1. de Bruin,EC,van de Pas,S.,Lips,EH,van Eijk,R.,van der Zee,MM,Lombaerts,M.,van Wezel,T.,Marijnen,CA,van Krieken,JH,Medema, JP,van de Velde,CJ,Eilers,PH和Peltenburg,LT(2005)。 宏观解剖与直肠癌的显微解剖:基质细胞对肿瘤细胞基因表达谱的微小影响。 a> BMC 6:142
  2. Wilming,SM,de Wilde,J.,Meijer,CJ,Berkhof,J.,Yi,Y.,van Wieringen,WN,Braakhuis,BJ,Meijer,GA,Ylstra,B.,Snijders,PJand Steenbergen,RD 2008)。 集成的基因组和转录谱可识别宫颈癌中基因表达改变的染色体基因座。 Genes Chromosomes Cancer 47(10):890-905。
  3. Wilming,SM,Snijders,PJ,Verlaat,W.,Jaspers,A.,van de Wiel,MA,van Wieringen,WN,Meijer,GA,Kenter,GG,Yi, R.,Meijer,CJ和Steenbergen,RD(2013)。 与染色体变化相关的改变的微小RNA表达有助于宫颈癌发生。 Onc ogene 32(1):106-116。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wilting, S. M. and Steenbergen, R. D. (2013). Total RNA Isolation after Laser-capture Microdissection of Human Cervical Squamous Epithelial Cells from Fresh Frozen Tissue . Bio-protocol 3(15): e848. DOI: 10.21769/BioProtoc.848.
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