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Optimizing Protocols for High-Quality RNA Extraction from Blood and Liver Tissues of the Broad-Snouted Caiman

Evelyn Cecilia López González, Lucía Magdalena Odetti, Gisela Laura Poletta, Nancy Denslow, Kevin J. Kroll, Pablo Ariel Siroski, María Virginia Parachú Marcó

Abstract


Transcriptomic information provides fundamental insights into biological processes and can be used to determine gene expression in cell, tissue, or organism under specific physiological conditions, or in response to any environmental perturbation. Extraction of high quality RNA is a challenging step mainly in non-traditional organisms, and protocols for preservation and isolation need to be adjusted in many cases. In the present work, we aimed to develop a protocol for preservation and isolation of high-quality and quantity of RNA from blood and liver tissues of Caiman latirostris. Three preservation methods were tested: 1) flash freezing (LN2) and storage at –80°C; 2) RNAlater® conservation with progressive cooling up to –80°C); 3) preservation in TRIzol® reagent, flash freezing in LN2 and storage at –80°C. Methods 1 and 2 were tested for liver, while 2 and 3 for blood. Our results showed that both preservation methods resulted in excellent outcomes for liver samples. For blood samples however, TRIzol® preservation was an efficient procedure for adequate RNA quality, quantity, and integrity, while conservation in RNAlater® solution was inadequate in both quality and quantity for an optimal RNA extraction. Appropriate protocols were established for each tissue and are being used now for transcriptomic studies in this sentinel organism.

Keywords


Crocodiles; Gene expression pattern; RNA conservation; RNA integrity; RNA isolation.

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References


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DOI: https://doi.org/10.30906/1026-2296-2021-28-4-197-204

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