Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Doreen Schwochow,Robert K Wayne,Laurel Ek Serieys,Olaf Thalmann

doi:10.1186/1472-6750-12-33

Abstract

BackgroundSince the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores.ResultsDespite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing.ConclusionBy carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.

Highlights

Since the emergence of generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations
We evaluated different RNA extraction methods applied to whole blood sampled under field conditions that resemble situations researchers face when investigating non-model organisms in their natural habitat
We used samples collected from 20 carnivores and RNA from each individual was preserved using five methods resulting in a total of 99 RNA extractions (Additional file 1: Figure S1, Additional file 1: Table S1)

Summary

Introduction

Since the emergence of generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. [1,2,3]), their molecular foundation remains largely unexplored opportunities have arisen to investigate the genetic mechanisms facilitating adaptation in natural populations [10,11,12,13]. The introduction of transcriptome sequencing via RNAseq [14,15] has led to important findings in the field of ecological genomics [16,17,18]. Owing to their high sensitivity, these novel technologies require special precautions in sample handling and preparation, a pre-requisite not often achievable for free-ranging, natural populations of vertebrates. Sampling via fat or muscle tissue biopsies may be possible in some exceptional cases, the primary least invasive and abundant source of RNA in vertebrates remains whole blood

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