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

LÓPEZ GONZÁLEZ EVELYN C.; SIROSKI PABLO ARIEL; ODETTI, LUCÍA; POLETTA, GISELA L.; PARACHÚ MARCÓ, MA. VIRGINIA; LÓPEZ GONZÁLEZ EVELYN C.; SIROSKI PABLO ARIEL; ODETTI, LUCÍA; PARACHÚ MARCÓ, MA. VIRGINIA; POLETTA, GISELA L.

Santa Fe

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

Crocodile Specialist Group (SSC-IUCN)/Universidad Nacional del Litoral

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 broadsnouted 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.