Optimized methods for obtaining sequencing-quality RNA from blubber of free-ranging cetaceans collected under field...

Cetaceans (whales, porpoises and dolphins) play critical roles in marine ecosystems, but many populations are declining and are vulnerable to anthropogenic disturbance. Understanding the impacts of disturbance on the physiology and health of cetacean populations and developing robust methods of assessing them are critical for their conservation. Many current approaches for studying stress in cetaceans do not address the downstream impacts of stress hormones and contaminants, which mediate their effects by altering gene activity in target tissues. The latter can be examined by transcriptome sequencing, which can rapidly produce species- and tissue-specific global gene expression profiles that can be correlated with hormone and contaminant levels to identify markers of stress and pollutant exposure. However, transcriptome studies of cetacean blubber have been limited by the high lipid and structural fibre content of this tissue, which typically yields low-quality RNA that is not suitable for sequencing. In this study, we conducted a comprehensive comparison of tissue handling and RNA extraction methods for transcriptome studies of blubber collected from free-ranging cetaceans under field conditions. We subsampled blubber biopsies obtained from wild bottlenose dolphins during routine health assessments and compared the effect of sample preservation, tissue homogenization and choice of nucleic acid extraction kit on RNA yield and integrity. We found that flash-freezing blubber upon collection, homogenization using cryogenic milling followed by bead beating and RNA extraction using a phenol-guanidine-chloroform and silica spin column kit designed for fatty and fibrous tissues significantly improve RNA quality. Using the pipeline that we developed, we show that it is possible to obtain large yields of intact RNA across the full depth of dolphin blubber with integrity values that exceed those reported thus far (up to 8.3) and that are suitable for stress biomarker discovery by RNA sequencing, facilitating health assessments of wild cetaceans sampled by remote biopsy.