Effect of RNA preservation methods on RNA quantity and quality of field-collected avian whole blood

Author:

Harvey Johanna A12ORCID,Knutie Sarah A1ORCID

Affiliation:

1. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, USA

2. Division of Invertebrate Zoology, American Museum of Natural History, New York, NY, USA

Abstract

The transcriptome comprises all RNA molecules in a sample, tissue, or organism. A limitation of comparative transcriptomic studies which compare gene expression between individuals often under some differing exposure or treatment, of wild avian populations continues to be sample preservation of high-quality RNA (i.e., ribonucleic acids that transfer, translate, and regulate the genetic code from DNA into proteins). Field sampling of wild bird blood provides challenges as RNA degradation progresses quickly, due in large part to the high nuclease content of avian blood and because cryopreservation is often not feasible at remote locations. The introduction of commercial buffers for preservation of RNA enables field-collected studies as these buffers deactivate nucleases which degrade target nucleic acids. We seek to compare the effectiveness of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at varying concentrations along with a dry ice-based flash freezing method to determine optimal preservation methods for field-collected avian blood samples. To determine optimal preservation methods, we assessed RNA quantity and quality metrics: RNA integrity numbers (RINe), rRNA ratios, and total RNA concentration. Nucleated red blood cells, a characteristic common across non-mammalian vertebrates, provide sufficient transcriptionally active material enabling the identification of potentially active gene regions from small and non-lethal samples (∼ 20 μl). A protocol was also optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of birds with a minimum threshold of 5 g body mass. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations provide sample protection from RNA degradation. We recommend that caution be exercised when using dry ice-based flash-freezing alone for sample preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial sample volumes.

Funder

Gerstner Family Foundation

University of Connecticut

American Ornithological Society

Publisher

SAGE Publications

Subject

Animal Science and Zoology,Ecology, Evolution, Behavior and Systematics

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