Abstract
BackgroundDescribing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 μm in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar).FindingsWe evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen).ConclusionsWe present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.
Highlights
Describing and evaluating miRNA inventories with Generation Sequencing is a goal of scientists from a wide range of fields
The AMR kit was best for extracting high quality totalRNA (RQI, RIN = 10) from single Gyrodactylus salaris individuals with a yield of 15.4 ng (Figure 1); Figure 1 1 Gyrodactylus salaris individual
Electropherogram (Experion) of totalRNA extracted from 1 Gyrodactylus salaris individual using the Phenol-free Total RNA Purification kit from Amresco (AMR). 1% of the extraction was loaded on an Experion RNA HighSens Chip
Summary
Describing and evaluating miRNA inventories with Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do contain abundant ribosomal RNAs but are enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 μm in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). In evolutionary terms miRNAs are unusual in that they are continuously added to, highly conserved, and rarely lost from metazoan genomes [13,14]. They are under strong selection, and may represent candidate phylogenetic markers. It may even be possible to reconstruct the miRNA complement of the last common ancestor of all Metazoa [15,16,17]
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