Optimizing Protocols for High-quality RNA Extraction from Blood and Liver Tissues of Caiman latirostris (Broad-snouted caiman)

EVELYN CECILIA LOPEZ GONZALEZ; LUCIA ODETTI; GISELA LAURA POLETTA; PABLO ARIEL SIROSKI; MARIA VIRGINIA PARACHÚ MARCÓ

Santa Fe

Congreso; 25th Working Meeting of the IUCN-SSC Crocodile Specialist Group; 2018

Universidad Nacional del Litoral - Proyecto Yacare

Transcriptomic information provides fundamental insights into biological processes and can be used to determine which genes are up- or down-regulated (as transcribed messenger RNA: mRNA) in cell, tissue, organ, or organism under specific physiological conditions or in response to an environmental perturbation, such as exposure to a toxic chemical. Extraction of high quality RNA is a challenging step mainly in non-traditional organisms, and protocols for preservation and extraction need to be adjusted in many cases. Our objective was to optimize preservation protocols for isolation of high-quality and quantity RNA from blood and liver tissues on broad-snouted caiman through the comparison of absorbance ratios and RNA integrity number (RIN) values to assess RNA quality. Four preservation treatments were tested: 1) flash freezing (N2 liquid) and storage at -80°C; 2) RNAlater® conservation with a progressive cooling (room temperature, refrigerator, storage at -20°C and storage at -80°C); 3) preservation in TRIzol® reagent and storage at -80°C and 4) direct extraction with TRIzol® from fresh cells. Our results showed higher RNA quality and quantity in liver than blood tissue in the four different preservation protocols. Moreover, RNAlater® conservation was inadequate for blood because of RNA degradation while liver tissue preserved in RNAlater® showed good quantity and quality of RNA but its concentration was higher with other preservation methods. TRIzol® treatment was the most efficient procedure for an adequate RNA quality, quantity and integrity in C. latirostris blood and liver tissues, both through immediately extraction or -80 °C conservation. This protocol was stablished for both tissues and is now being used for transcriptomic studies in both tissues.