MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits
Background Describing 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...
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ftoslouniv:oai:www.duo.uio.no:10852/46519 2023-05-15T15:32:03+02:00 MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits Fromm, Bastian David Harris, Philip Bachmann, Lutz 2011 http://hdl.handle.net/10852/46519 http://urn.nb.no/URN:NBN:no-50730 https://doi.org/10.1186/1756-0500-4-217 eng eng http://urn.nb.no/URN:NBN:no-50730 BMC Research Notes. 2011 Jun 29;4(1):217 http://hdl.handle.net/10852/46519 http://dx.doi.org/10.1186/1756-0500-4-217 URN:NBN:no-50730 Fulltext https://www.duo.uio.no/bitstream/handle/10852/46519/1/13104_2011_Article_972.pdf Fromm et al; licensee BioMed Central Ltd. Attribution 2.0 Generic http://creativecommons.org/licenses/by/2.0/ CC-BY Journal article Tidsskriftartikkel Peer reviewed PublishedVersion 2011 ftoslouniv https://doi.org/10.1186/1756-0500-4-217 2020-06-21T08:48:52Z Background Describing 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). Findings We 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). Conclusions We 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. Article in Journal/Newspaper Atlantic salmon Salmo salar Universitet i Oslo: Digitale utgivelser ved UiO (DUO) BMC Research Notes 4 1 |
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Universitet i Oslo: Digitale utgivelser ved UiO (DUO) |
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English |
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Background Describing 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). Findings We 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). Conclusions We 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. |
format |
Article in Journal/Newspaper |
author |
Fromm, Bastian David Harris, Philip Bachmann, Lutz |
spellingShingle |
Fromm, Bastian David Harris, Philip Bachmann, Lutz MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
author_facet |
Fromm, Bastian David Harris, Philip Bachmann, Lutz |
author_sort |
Fromm, Bastian |
title |
MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
title_short |
MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
title_full |
MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
title_fullStr |
MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
title_full_unstemmed |
MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits |
title_sort |
microrna preparations from individual monogenean gyrodactylus salaris-a comparison of six commercially available totalrna extraction kits |
publishDate |
2011 |
url |
http://hdl.handle.net/10852/46519 http://urn.nb.no/URN:NBN:no-50730 https://doi.org/10.1186/1756-0500-4-217 |
genre |
Atlantic salmon Salmo salar |
genre_facet |
Atlantic salmon Salmo salar |
op_relation |
http://urn.nb.no/URN:NBN:no-50730 BMC Research Notes. 2011 Jun 29;4(1):217 http://hdl.handle.net/10852/46519 http://dx.doi.org/10.1186/1756-0500-4-217 URN:NBN:no-50730 Fulltext https://www.duo.uio.no/bitstream/handle/10852/46519/1/13104_2011_Article_972.pdf |
op_rights |
Fromm et al; licensee BioMed Central Ltd. Attribution 2.0 Generic http://creativecommons.org/licenses/by/2.0/ |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.1186/1756-0500-4-217 |
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BMC Research Notes |
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4 |
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1 |
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1766362553546440704 |