Buenfil-Rojas Asela Marisol, González-Jáuregui Mauricio, Ochiai Mari, Iwata Hisato
Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime, Japan.
Crocodile Research and Management for Conservation, Carr. San Agustín Km 4.5, 24522, San Francisco de Campeche, Campeche, Mexico.
MethodsX. 2025 Apr 15;14:103315. doi: 10.1016/j.mex.2025.103315. eCollection 2025 Jun.
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 , 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.•This study optimizes non-invasive RNA extraction from crocodile scutes, enabling conservation research and transcriptomic analysis.
应对从野生动物硬组织中提取RNA的挑战至关重要，特别是考虑到物种保护以及避免采用致死性采样方法的伦理要求。本研究聚焦于优化从墨西哥尤卡坦半岛特有物种的尾盾进行非侵入性RNA提取的方案，突出了在实验室条件有限的热带地区开展研究的重要性。在使用RNA保存缓冲液处理盾片组织的同时，我们探索了各种组织破碎和匀浆技术，以促进RNA的分离和纯化。通过评估RNA的纯度和完整性来确定最佳提取方法。优化后的方案包括对75毫克样品进行超声处理，随后进行3小时的蛋白酶K孵育，得到的RNA浓度为18.7至154.7纳克/微升，纯度令人满意（260/280比值约为2），完整性良好（RNA完整性数值>5.5）。通过定量PCR分析进一步验证，证实了提取的RNA适用于基因表达水平研究，并且足以用于下一代测序（NGS）。该方案可为开发针对其他具有硬组织的非模式物种的类似方法提供基础。•本研究优化了从鳄鱼盾片中进行非侵入性RNA提取的方法，可用于保护研究和转录组分析。
