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

ABSTRACT A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, −109°C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RIN e ), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples 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 samples volumes and carryover across processing steps. LAY SUMMARY Preserving RNA samples collected in field conditions, under extreme or variable conditions, remains a challenge and continues to limit the study of gene expression in wild birds. We optimized RNA extractions for small-volume whole avian blood samples. We compared commonly used RNA preservations methods, commercial RNA buffers at varying concentrations along with a dry ice-based flash freezing method, for field collected blood bird samples. All RNA buffers preserved samples resulted in high quality RNA extractions suitable for downstream RNA sequencing, while dry ice-based flash freezing resulted in less reliable sample preservation. Our results support the feasibility of blood sampling as a method of non-lethal sampling that will enable the increased usage RNA sequencing in wild bird studies. KEY TERMS RNA-Seq high-throughput sequencing which profiles RNA molecules in a sample at the collection timepoint. Provides content and abundance information and may identify novel genes and isoforms. Prior to sequencing, library preparation may either target mRNA or total RNA with the use of enrichment or depletion steps. Transcriptome all RNA transcripts, both coding and non-coding, in an individual. rRNA ratio (28S/18S) commonly used ratio of 28S and 18S ribosomal RNA (rRNA) (band sizes or peak area ratio) that informs the integrity of RNA. Appropriate ratios vary by taxonomic groups and associated rRNA sizes. Acceptable ratios of 28S to 18S are approximately 2.0 for eukaryotes. Electropherogram a quantitative distribution of fragment size. Qubit 4 Fluorometer fluorescence-based quantification of RNA molecule concentration using fluorescent intercalating dyes which bind to target molecules. Agilent 4200 TapeStation high-throughput analyzer which uses microfluidics, microcapillary electrophoresis, and fluorescence detection that allows for size determination of the isolated molecules. Used for quality control and quantification of DNA and RNA samples for downstream methods. Reports metrics to assess sample (DNA, RNA, protein) integrity and quantification, which includes: electropherogram, gel-like image, concentration values, 28S/18S rRNA ratios, and an algorithmically determined integrity score (RIN e ). Provides a more complete characterization of the measured samples then previous analyzers for downstream analysis. Fast zone area between small RNAs and the 18S rRNA fragment (~200 bp to 1.8 kb). The fast zone is where the degradation of the 18S and 28S peaks accumulates. RIN e RNA integrity number (RIN equivalent) which is determined by quantitative measurements of total RNA degradation. Measurements are based on features of the electropherogram which assesses degradation or lack of degradation by measuring the ratio of area in the fast zone to the 18S peak signal. RIN of 10 signifies the highest quality intact RNA and zero is the worst score indicates a low-quality degraded sample.