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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, 10412 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.

材料与试剂

 

1.       石蜡包埋标本

2.       硅化管(Thomas Scientific, 2591L12)

3.       100%二甲苯(THERMO SCIENTIFIC, 6601)

4.       100% 乙醇

5.       蛋白酶K及消化缓冲液(Ambion cat# AM2546)

6.       RNA抽提试剂Trizol (Invitrogen,15596-018)

7.       氯仿  (Sigma, C2432)

8.       糖原 (Roche Applied Science, 10901393001)

9.       异丙醇(Isopropanol)

10.    RNase-free 水或TE 缓冲液 (USB, 75834, Promega P1193)

11.    Tris-HCl (Invitrogen, 15568-025)

12.    CaCl2 (Sigma, C5670)

13.    十二烷基硫酸钠(Invitrogen, 15525-017)

 

设备

 

1.       显微镜用薄片切片机

2.       微量离心机

 

实验步骤

 

1.       用显微镜用薄片切片机从石蜡块内部切下20 μm组织切片, 尽量减少储存过程暴露在大气中对核酸造成的损坏.

(回收miRNA,建议切片 >=20 μm,否则在脱蜡漂洗时miRNA 将损失)

2.       组织切片放入1.5 ml的硅化管,样品中加入1 ml 100%二甲苯。在50孵育3分钟,以融化石蜡。样品以最大转速离心以将组织颗粒化,之后在不干扰颗粒的条件下弃去二甲苯. 重复用二甲苯洗一次。

3.       1 ml 100%乙醇漂洗所得颗粒两次并封干。

4.       150μl 1X蛋白酶K消化缓冲液(含有500 μg/ml的蛋白酶K)加入到每个样品中,在55孵育3小时。

5.       加入1 ml Trizol (Invitrogen) 到每个样品中,在15 30 °C孵育至少5min以分离核蛋白复合物。加入0.2 ml氯仿, 有力震荡管15 s,并在15 °C 30 °C 孵育2-3分钟。在转速不超过12,000 x g条件下4 °C.离心样品15 min

6.       将水相转移至新管中,加入10 μg 糖原并混合。0.6ml异丙醇混合沉淀总RNA, 并把管放在 -20 °C 至少1 hr. 2~8 °C  12,000 x g离心10 min

7.       100%的乙醇洗涤RNA的颗粒,简单晾干。用RNase-free水或TE溶解。

注意:

大多数成熟 microRNA和其他小RNA都完好无损,而大多数的mRNA和其他长RNA在福尔马林固定中破碎。产量比一个商业试剂盒(Ambion, Austin, TX)3倍。

RNA产物适合于microRNA研究或mRNA3SEQ分析。

硅化管和糖原,用以获得更高的小RNA的产量.

 

配方

 

1.       蛋白酶消化缓冲液

20 mM Tris-HCl, pH 8.0

1 mM CaCl2,

0.5%十二烷基硫酸钠(SDS

 

参考文献

 

1.        Ma Z., Lui W.O., Fire A., Dadras S.S. (2009). Profiling and discovery of novel miRNAs from formalin-fixed, paraffin-embedded melanoma and nodal specimens. J Mol Diagn 11(5): 420-9. 

 

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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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