title: Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
abstract: There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.
authors.affiliation: Physiology Weihenstephan and ZIEL Research Center for Nutrition and Food Sciences, Technical University Munich, Freising, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Spornraft
authors.affiliation: Physiology Weihenstephan and ZIEL Research Center for Nutrition and Food Sciences, Technical University Munich, Freising, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Kirchner
authors.affiliation: Genomics Core Facility, EMBL European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Haase
authors.affiliation: Genomics Core Facility, EMBL European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Benes
authors.affiliation: Physiology Weihenstephan and ZIEL Research Center for Nutrition and Food Sciences, Technical University Munich, Freising, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
authors.affiliation: Physiology Weihenstephan and ZIEL Research Center for Nutrition and Food Sciences, Technical University Munich, Freising, Germany.
authors.forename: Irmgard
authors.initials: I
authors.lastname: Riedmaier
mesh_terms: D000818:Animals; D002417:Cattle; D019295:Computational Biology; D015723:Gene Library; D058977:Molecular Sequence Annotation; D059372:Nucleotide Motifs; D012313:RNA / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D058727:RNA, Small Untranslated / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D017423:Sequence Analysis, RNA
chemical_list: D058727:RNA, Small Untranslated; D012313:RNA