A comparative study of RNA yields from museum specimens, including an optimized protocol for extracting RNA from formalin-fixed specimens

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens with the downstream goal of surveying for zoonotic diseases, and may also offer the opportunity to examine changes in host gene expression. We provide RNA quality and quantity measures for specimens from five bat genera collected in China and Myanmar from 1886 to 2003. The specimens were preserved in a variety of ways including formalin-fixed ethanol-preserved, ethanol-preserved (stored at room temperature or frozen), and flash frozen without buffer. We find that RNA extracted from museum specimens is highly fragmented, but usable for whole genome short-read sequencing and targeted amplification with qPCR. Formalin-fixed ethanol-preserved specimens yield similar quality of RNA to FFPE samples, and quality is sufficient for qPCR screening for specific primers and probes. This represents an extension of the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.