MicroRNAs (miRNAs) are key regulators of many biological processes in eukaryotes . Besides their investigation through bioinformatical analyses of whole genomes, research focuses on analyses of miRNAs from whole organisms, specific tissues, and/or developmental stages without a fully sequenced and annotated genome at hand .
MiRNAs are single-stranded, 22 nucleotide long, noncoding transcripts derived from different genome-encoded hairpin precursors, and regulate gene expression by various mechanisms . First described from Caenorhabditis elegans they represent the most recently discovered gene regulators, involved in a broad variety of biological processes including cell proliferation and metabolism , developmental timing , cell death , haematopoiesis , neuron development , tumorigenesis , DNA methylation and chromatin modification , and as immune defense against viruses . In evolutionary terms miRNAs are unusual in that they are continuously added to, highly conserved, and rarely lost from metazoan genomes [13, 14]. Clearly they are under strong selection, and may therefore represent candidate phylogenetic markers. It may even be possible to reconstruct the miRNA complement of the last common ancestor of all Metazoa [15–17].
Several methods for isolation of totalRNA have been developed, with the focus primarily on high molecular weight RNAs . Commercially available totalRNA kits are affordable, fast, and suitable for RNA extraction from a broad spectrum of samples. Most manufacturers promote their products as suitable for extraction of totalRNA, but frequently it is unclear whether they are equally suitable for smallRNA species, and especially for miRNAs. For extractions of these molecules not only is overall RNA quality and integrity an issue, but also due to their low abundance, yield is of high importance. This is a particular issue if only limited material is available, as in the case of, for example, micro-dissected tissue samples or small invertebrates.
The current study focuses on the ectoparasitic platyhelminth Gyrodactylus salaris (Monogenea, Gyrodactylidae). This parasite is responsible for a major epidemic disease of wild salmon in Norway and Russia , but gyrodactylids are widespread on teleost fishes and several cause disease in aquaculture [19, 20]. Apart from this applied importance, an understanding of the miRNA complement of gyrodactylids will contribute to our understanding of platyhelminth, and early metazoan evolution. However, the individual parasites are only 500 μm in length, presenting a challenge for RNA extraction methodologies. In this study we tested 6 commercially available totalRNA extraction kits for their performance when using as few as 1, 10 and 100 Gyrodactylus salaris individuals respectively. Particular emphasis was placed on assessing (i) total RNA yield (ii) RNA integrity (iii) smallRNA yield, and (iv) miRNA yield.