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

Abstract

Addressing the challenge of RNA extraction from hard tissues of wild animals is crucial, especially given the species' conservation and the ethical imperative to avoid lethal sampling methods. This study focuses on optimizing a protocol for non-invasive RNA extraction from the caudal scutes of Crocodylus moreletii, an endemic species in the Yucatan Peninsula, Mexico, highlighting the significance of conducting research in tropical areas with limited laboratory access. Accompanying with RNA preservation buffer for the scute tissue, we explored various tissue disruption and homogenization techniques to facilitate RNA isolation and purification. The purity and integrity of RNA were assessed to determine the best extraction method. The optimized protocol involved ultrasonication of 75 mg samples, followed by a 3-hour Proteinase K incubation, yielding RNA with concentrations from 18.7 to 154.7 ng/µL, satisfactory purity (260/280 ratio ∼2), and integrity (RNA Integrity Number >5.5). Further validation through quantitative PCR analyses confirmed the suitability of the extracted RNA for studies on gene expression levels and were sufficient for next-generation sequencing (NGS). This protocol may provide a basis for developing similar methodologies for other non-model species with hard tissues.

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  This study optimizes non-invasive RNA extraction from crocodile scutes, enabling conservation research and transcriptomic analysis.