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Related Experiment Videos

Protecting RNA in fixed tissue: an alternative method for LCM users.

Alexandre Hiroaki Kihara1, Anselmo Sigari Moriscot, Paula Juliana Ferreira

  • 1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 São Paulo, SP, Brazil. kihara@icb.usp.br

Journal of Neuroscience Methods
|July 20, 2005
PubMed
Summary

This study presents an improved protocol for laser capture microdissection (LCM) to preserve RNA integrity and tissue morphology. The optimized method enhances RNA yield and quality, making it a reliable alternative for researchers.

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Area of Science:

  • Molecular Biology
  • Histology
  • Biotechnology

Background:

  • RNA degradation is a significant challenge in techniques combining RNA integrity and morphology preservation, like laser capture microdissection (LCM) followed by RT-PCR.
  • Commercially available RNA isolation kits for LCM samples are often prohibitively expensive.

Purpose of the Study:

  • To develop an accessible protocol that maintains acceptable tissue morphology.
  • To facilitate precise laser capture of selected cells.
  • To enhance RNA yield and quality from LCM samples.

Main Methods:

  • Retinas were dissected, briefly treated with an RNA preservative, and fixed in 2% paraformaldehyde.
  • Cryostat sectioning was performed, followed by laser capture microdissection (LCM) on retinal sections.

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  • RNA isolation was conducted using a modified TRIzol protocol, and RNA quality/yield were assessed via real-time PCR.

    • Main Results:

    • The RNA preservative and rapid fixation protocol preserved tissue morphology, enabling accurate cell microdissection.
    • Minor modifications to the standard RNA isolation procedure significantly improved RNA yield and quality.
    • Only samples processed with the described protocol yielded amplifiable mRNA, confirmed by real-time PCR.

    Conclusions:

    • The combined protocol offers a reliable and cost-effective alternative for researchers using LCM.
    • This method successfully addresses the limitations of RNA degradation and high costs associated with LCM sample preparation.
    • The optimized protocol ensures high-quality RNA suitable for downstream applications like RT-PCR.