miRNA in situ hybridization in formaldehyde and EDC-fixed tissues

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MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues
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MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Published on: November 30, 2013

John T G Pena1, Cherin Sohn-Lee, Sara H Rouhanifard

  • 1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, New York 10065, USA.

Nature Methods
|January 13, 2009
PubMed

Insights

Formaldehyde fixation causes microRNA loss during in situ hybridization (ISH). Using a carbodiimide fixative prevents this loss, preserving microRNAs for accurate analysis in mouse tissues.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • MicroRNAs are crucial regulatory molecules involved in numerous biological processes.
  • Dysregulation of microRNAs is linked to various diseases, making their accurate detection important.
  • Standard in situ hybridization (ISH) techniques can lead to significant loss of microRNAs from tissue samples.

Purpose of the Study:

  • To investigate the impact of fixation methods on microRNA integrity during ISH.
  • To identify a fixation method that preserves microRNAs for improved detection.
  • To optimize hybridization conditions for enhanced microRNA detection using locked nucleic acid probes.

Main Methods:

  • Comparison of formaldehyde fixation versus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide fixation for mouse tissue sections.
  • In situ hybridization (ISH) utilizing locked nucleic acid (LNA) probes.
  • Monitoring of nucleic acid melting temperatures to determine optimal hybridization parameters.

Main Results:

  • Conventional formaldehyde fixation leads to substantial loss of microRNAs from mouse tissue sections.
  • Fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide effectively prevents microRNA loss by immobilizing them.
  • Optimal hybridization parameters were established for 130 LNA probes based on melting temperature analysis.

Conclusions:

  • The choice of fixation method significantly impacts microRNA yield in ISH.
  • Carbodiimide fixation offers a superior alternative to formaldehyde for preserving microRNA integrity.
  • Optimized ISH protocols enhance the reliability of microRNA detection in biological samples.

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Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...