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Total RNA is extracted from fixed biological specimens by this method with higher yield than commercial kits. The product contains intact micro RNAs and small RNAs, and fragmented long RNAs.

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Total RNA Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Blocks
从福尔马林固定的石蜡包埋(FFPE)模块中提取总 RNA

分子生物学 > RNA > RNA 提取
作者: Zhihai Ma
Zhihai MaAffiliation: Department of Pathology, Stanford University School of Medicine, Stanford, USA
For correspondence: zhihaima@stanford.edu
Bio-protocol author page: a42
Vol 2, Iss 7, 4/5/2012, 11907 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.161

[Abstract] Total RNA is extracted from fixed biological specimens by this method with higher yield than commercial kits. The product contains intact micro RNAs and small RNAs, and fragmented long RNAs.

[Abstract] 此方法从固定的生物标本中提取总RNA,比商业试剂盒具有更高的产量。其产物含有完整的micro RNA,小RNA,以及片段状的长RNA

Materials and Reagents

  1. Paraffin embedded specimen
  2. 100% xylene (Thermo Fisher Scientific, catalog number: 6601 )
  3. 100% ethanol
  4. Protease K (Life Technologies, Ambion®, catalog number: AM2546 )
  5. Trizol (Life Technologies, Invitrogen™, catalog number: 15596-018 )
  6. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  7. Glycogen (F. Hoffmann-La Roche, catalog number: 10901393001 )
  8. Isopropyl alcohol (Isopropanol)
  9. RNase-free water or TE buffer (USB, catalog number: 75834 ; Promega Corporation, catalog number: P1193 )
  10. Tris-HCl (Life Technologies, Invitrogen™, catalog number: 15568-025 )
  11. CaCl2 (Sigma-Aldrich, catalog number: C5670 )
  12. Sodium dodecyl sulfate (Life Technologies, Invitrogen™, catalog number: 15525-017 )
  13. Commercial kit (Life Technologies, Ambion®, Austin, TX)
  14. Sodium dodecyl sulfate
  15. Protease digestion buffer (see Recipes)

Equipment

  1. Microtome
  2. Microcentrifuge
  3. Siliconized tubes (Thomas Scientific, catalog number: 2591L12 )

Procedure

  1. Cut 20 μm sections from the interior of the paraffin block using a microtome, to minimize the nucleic acid damaged by exposure to the atmosphere during storage (for recovery of miRNA ≥20 μm slices are recommended, otherwise the miRNA will be lost during deparaffin washes).
  2. Place tissue slices into 1.5 ml siliconized tubes, and add 1 ml 100% xylene to the sample.
  3. Incubate at 50 °C for 3 min to melt the paraffin. Centrifuge the sample for 1 min at maximum speed to pellet the tissue, then discard the xylene without disturbing the pellet. Repeat the xylene wash once.
  4. Wash the pellet twice with 1 ml 100% ethanol and air dry.
  5. Add 150 μl 1x protease K digestion buffer containing 500 μg/ml protease K to each sample, incubate at 55 °C for 3 h.
  6. Add 1 ml Trizol to each sample, incubate at 15 to 30 °C for at least 5 min to dissociate nucleoprotein complexes. Add 0.2 ml of chloroform, vortex the tubes vigorously for 15 sec and incubate at 15 °C to 30 °C for 2 to 3 min. Centrifuge the samples at no more than 12,000 x g for 15 min at 4 °C.
  7. Transfer the aqueous phase to a fresh tube, add 10 μg glycogen and mix. Precipitate the total RNA by mixing with 0.6 ml isopropyl alcohol, and put the tube at -20 °C for at least 1 h. Centrifuge at 12,000 x g for 10 min at 2-8 °C.
  8. Wash the RNA pellet with 100% ethanol, briefly air-dry. Dissolve in RNase-free water or TE.

Notes

  1. Most mature microRNAs and other small RNAs are intact, while most mRNA and other long RNAs are fragmented during formalin fixation. The yield is about 3 times higher than a commercial kit.
  2. The RNA product is suitable for microRNA study or mRNA profiling by 3SEQ.

Recipes

  1. Protease digestion buffer
    20 mM Tris-HCl (pH 8.0)
    1 mM CaCl2
    0.5% sodium dodecyl sulfate

Acknowledgments

This protocol was adapted from Ma et al. (2009).

References

  1. Ma, Z., Lui, W. O., Fire, A. and Dadras, S. S. (2009). Profiling and discovery of novel miRNAs from formalin-fixed, paraffin-embedded melanoma and nodal specimens. J Mol Diagn 11(5): 420-429.

材料和试剂

  1. 石蜡嵌入样品
  2. 100%二甲苯(Thermo Fisher Scientific,目录号:6601)
  3. 100%乙醇
  4. 蛋白酶K(Life Technologies,Ambion ,目录号:AM2546)
  5. Trizol(Life Technologies,Invitrogen TM,目录号:15596-018)
  6. 氯仿(Sigma-Aldrich,目录号:C2432)
  7. 糖原(F.Hoffmann-La Roche,目录号:10901393001)
  8. 异丙醇(异丙醇)
  9. 无RNase水或TE缓冲液(USB,目录号:75834; Promega Corporation,目录号:P1193)
  10. Tris-HCl(Life Technologies,Invitrogen TM,目录号:15568-025)
  11. CaCl 2(Sigma-Aldrich,目录号:C5670)
  12. 十二烷基硫酸钠(Life Technologies,Invitrogen TM,目录号:15525-017)
  13. 商业试剂盒(Life Technologies,Ambion ,Austin,TX)
  14. 十二烷基硫酸钠
  15. 蛋白酶消化缓冲液(参见配方)

设备

  1. 切片机
  2. 微量离心机
  3. 硅化管(Thomas Scientific,目录号:2591L12)

程序

  1. 使用切片机从石蜡块的内部切割20μm切片,以最小化在储存期间暴露于大气损伤的核酸(为了恢复miRNA≥20μm切片,否则miRNA将在脱蜡洗涤期间丢失) 。
  2. 将组织切片放入1.5毫升硅化试管,并加入1毫升100%二甲苯的样品。
  3. 在50℃下孵育3分钟以融化石蜡。以最大速度离心样品1分钟以沉淀组织,然后丢弃二甲苯而不干扰沉淀。重复二甲苯清洗一次。
  4. 用1ml 100%乙醇洗涤沉淀两次并风干
  5. 向每个样品中加入150μl含有500μg/ml蛋白酶K的1x蛋白酶K消化缓冲液,在55℃孵育3小时。
  6. 向每个样品中加入1ml Trizol,在15至30℃孵育至少5分钟以解离核蛋白复合物。加入0.2ml氯仿,剧烈涡旋管15秒,并在15℃至30℃温育2至3分钟。在4℃下将样品以不超过12,000×g离心15分钟。
  7. 将水相转移到新鲜管中,加入10μg糖原并混合。通过与0.6ml异丙醇混合沉淀总RNA,并将管在-20℃至少1小时。在2-8℃下以12,000×g离心10分钟
  8. 用100%乙醇洗涤RNA沉淀,短暂空气干燥。溶于无RNase的水或TE

笔记

  1. 大多数成熟的微小RNA和其他小RNA是完整的,而大多数mRNA和其他长RNAs在福尔马林固定期间片段化。 产率是商业试剂盒的约3倍。
  2. RNA产物适用于通过3SEQ的微RNA研究或mRNA谱。

食谱

  1. 蛋白酶消化缓冲液
    20mM Tris-HCl(pH8.0) 1mM CaCl 2
    0.5%十二烷基硫酸钠

致谢

该协议改编自Ma等人(2009)。

参考文献

  1. Ma,Z.,Lui,W.O.,Fire,A。和Dadras,S.S。(2009)。 从福尔马林固定,石蜡包埋的黑素瘤和淋巴结标本中分析和发现新型miRNA。 a> J Mol Diagn 11(5):420-429。
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How to cite this protocol: Ma, Z. (2012). Total RNA Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Blocks. Bio-protocol 2(7): e161. DOI: 10.21769/BioProtoc.161; Full Text



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6/7/2012 5:17:05 AM  

Dear Sirs

My name is Masood; PhD student at the University of Saarland. I'd really appreciate it if you could tell me in which step you recommend me to digest the genomic DNA contamination?

because I would like to do qPCR for both miRNAs and mRNA.

In advance, thank you for your time and consideration and I look forward to hearing from you.
Sincerely yours
Abu-Halima, Masood

University of Saarland
Human Genetics Dep. 60
Kirrberger Stra?e 100
66421 Homburg Saar
Tel: +49(0)17671665654
E-Mail: masood@daad-alumni.de
Germany

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