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Laser capture microdissection for transcriptomic profiles in human skin biopsies.

Silvia Santoro1, Ignazio Diego Lopez2, Raffaella Lombardi3

  • 1Laboratory of Human Genetics of Neurological Disorders, Institute of Experimental Neurology (INSPE), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.

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|June 21, 2018
PubMed
Summary

We optimized a protocol for extracting high-quality RNA from human skin biopsies, enabling reliable RNA sequencing data for research. This method addresses challenges like nucleases and RNA instability in skin tissue.

Keywords:
Idiopathic neuropathyLaser capture microdissectionRNA sequencingSkin biopsyTranscriptomics

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Area of Science:

  • Molecular Biology
  • Genomics
  • Dermatology

Background:

  • Acquiring reliable tissue-specific RNA sequencing data from human skin biopsies is crucial for research.
  • Challenges include isolating specific skin layers, high nuclease activity, and RNA instability.
  • Existing methods require optimization for consistent, high-quality RNA extraction.

Purpose of the Study:

  • To develop and optimize a protocol for extracting RNA from specific layers of human skin biopsies.
  • To ensure satisfactory quality and quantity of mRNA for sequencing.
  • To overcome methodological challenges in skin tissue RNA isolation.

Main Methods:

  • Optimized protocol involving collection, embedding, freezing, and histological staining of fresh-frozen human skin biopsies (14 subjects).
  • Incorporated RNA preservation (RNALater® Solution), temperature control, RNase Inhibitors, and reduced staining time.
  • RNA quality assessed by DV200; library preparation using TruSeq® RNA Access Kit; sequencing on HiSeq® 2500.

Main Results:

  • The optimized protocol successfully preserved RNA integrity in skin biopsy layers.
  • DV200 values indicated suitability for library preparation, outperforming RNA Integrity Number (RIN) for this application.
  • Generated adequate quality RNA sequencing data for downstream transcriptomic analysis.

Conclusions:

  • The implemented protocol enables reliable transcriptomics data generation from skin tissues.
  • The protocol is potentially applicable to other tissue types.
  • An initial preservation step allows for sample shipment, facilitating multicenter studies.