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

Abstract

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 in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

Century-old chromatin architecture preserved with formaldehyde

“…Precedents have been set by successful research on clinical FFPE samples [28], and a single study has recently retrieved RNA from formaldehyde-fixed museum specimens [29]. A better understanding of how fixation modulates the molecular signal in these contexts could revolutionise our ability to study long-term temporal trends in gene regulation.…”

The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation

“…Working with museum specimens [the burgeoning field of "museomics"; 181] is another promising avenue of research allowing to access to past amphibian biodiversity. However, there are several additional challenges associated with DNA degradation, preservation methods, and contamination that need to be overcome [182][183][184]. This is particularly relevant for wet-preserved amphibian specimens, as retrieving DNA can be challenging due to the often unknown fixation and preservation methods that can alter nucleotide integrity.…”

Temporal collections to study invasion biology

“…Further, epigenetic modifications can mediate environmental effects on phenotypes through gene expression and historical epigenomics may therefore allow ancestral patterns of gene expression to be inferred (e.g., Hahn et al., 2023; Rubi et al., 2020). These analyses could be coupled with direct reconstruction of gene expression profiles from natural history specimens (e.g., Marmol‐Sanchez et al., 2023), which is now possible, thanks to the development of protocols for extracting RNA from preserved specimens (e.g., Speer et al., 2022). DNA methylation patterns have already been investigated in ancient and historical specimens (e.g., Gokhman et al., 2014; Gokhman et al., 2016; Hahn et al., 2020; Niiranen et al., 2022; Orlando et al., 2015; Pedersen et al., 2014; Rubi et al., 2020; Smith et al., 2014), indicating that this approach holds promise for unravelling the role of heritable epigenetic modifications in facilitating rapid adaptation to environmental shifts.…”