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Developing an efficient protocol for RNA extraction from Morelet's crocodile caudal scute biopsies.

Asela Marisol Buenfil-Rojas1,2, Mauricio González-Jáuregui3,2, Mari Ochiai1

  • 1Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime, Japan.

Methodsx
|May 7, 2025
PubMed
Summary

We developed a non-invasive RNA extraction method from crocodile scutes. This protocol enables conservation research and transcriptomic analysis in hard-to-sample wild species.

Keywords:
CrocodyliansKeratinized tissuesRNA Extraction from Morelet's Crocodile Caudal Scute BiopsiesRNA purificationTranscriptomics

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Area of Science:

  • Conservation Biology
  • Molecular Biology
  • Wildlife Research

Background:

  • RNA extraction from hard tissues of wild animals presents challenges, especially for conservation efforts requiring non-lethal methods.
  • Research in tropical areas often faces limited laboratory infrastructure, necessitating robust and adaptable protocols.
  • Caudal scutes of *Crocodylus moreletii* were chosen as a source for non-invasive RNA sampling.

Purpose of the Study:

  • To optimize a non-invasive RNA extraction protocol from the caudal scutes of *Crocodylus moreletii*.
  • To assess RNA purity and integrity for downstream applications like gene expression studies.
  • To provide a foundation for similar methods applicable to other non-model species with hard tissues.

Main Methods:

  • Exploration of various tissue disruption and homogenization techniques.
  • Optimization of RNA isolation and purification using an RNA preservation buffer.
  • Ultrasonication of 75 mg samples followed by a 3-hour Proteinase K incubation.

Main Results:

  • Achieved RNA concentrations ranging from 18.7 to 154.7 ng/µL.
  • Obtained satisfactory RNA purity with a 260/280 ratio of approximately 2.
  • Confirmed RNA integrity with an RNA Integrity Number (RIN) greater than 5.5.
  • Validated RNA suitability for quantitative PCR and next-generation sequencing (NGS).

Conclusions:

  • The optimized protocol provides a reliable method for non-invasive RNA extraction from crocodile scutes.
  • The extracted RNA is suitable for gene expression analysis and transcriptomic studies.
  • This methodology can be adapted for RNA extraction from hard tissues of other non-model wild species, aiding conservation research.