A mammalian messenger RNA sex determination method from humpback whale (Megaptera novaeangliae) blubber biopsies

The large size of free-ranging mysticetes, such as humpback whales (Megaptera novaeangliae), make capture and release health assessments unfeasible for conservation research. However, individual energetic condition or reproductive health may be assessed from the gene expression of remotely biopsied tissue. To do this, researchers must reliably extract RNA and interpret gene expression measurements within the context of an individual's sex. Here, we outline an RNA extraction protocol from blubber tissue and describe a novel mammalian RNA sex determination method. Our method consists of a duplex reverse transcription-quantitative (real-time) polymerase chain reaction (RT-qPCR) with primer sets for a control gene (ACTB) and the X-chromosome inactivation gene (XIST). Products of each RT-qPCR had distinct melting temperature profiles based on the presence (female) or absence (male) of the XIST transcript. Using high-resolution melt analysis, reactions were sorted into one of two clusters (male/female) based on their melting profiles. We validated the XIST method by comparing results with a standard DNA-based method. With adequate quantities of RNA (minimum of approx. 9 ng µl^-1), the XIST sex determination method shows 100% agreement with traditional DNA sex determination. Using the XIST method, future cetacean health studies can interpret gene expression within the context of an individual's sex, all from a single extraction.