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Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
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Genetics
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JoVE Journal Genetics
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

DOI:
10.3791/57471-v

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12:24 min

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May 05, 2018

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, , ,
1Department of Research,Hackensack University Medical Center, 2Department of Medical Sciences,Seton Hall University, 3Department of Epidemiology and Population Health,Albert Einstein College of Medicine, 4Department of Nephrology and Hypertension,Hadassah – Hebrew University Medical Center

Chapters

  • 00:04Title
  • 00:393′ Barcoded Adapter Ligations with 18 Individual RNA Samples
  • 02:00Ligated Small RNA Purification
  • 05:265′ Ligated and 3′ Barcoded Purified Small RNAs Reverse Transcription
  • 08:12Pilot PCR and Large-scale PCR Amplification
  • 10:46Results: Protocol Robust Whether Fresh, Frozen, or Aged
  • 12:01Conclusion

Summary

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Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

Tags

Keywords: MicroRNASequencingCDNA LibraryFormalin-fixed Paraffin-embeddedRNAProtocolLigationAdapterPrecipitationCentrifugation

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