title: Influence of next-generation sequencing and storage conditions on miRNA patterns generated from PAXgene blood.
abstract: Whole blood derived miRNA signatures determined by Next-Generation Sequencing (NGS) offer themselves as future minimally invasive biomarkers for various human diseases. The PAXgene system is a commonly used blood storage system for miRNA analysis. Central to all miRNA analyses that aim to identify disease specific miRNA signatures, is the question of stability and variability of the miRNA profiles that are generated by NGS. We characterized the influence of five different conditions on the genome wide miRNA expression pattern of human blood isolated in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three individuals. The blood was subjected to different numbers of freeze/thaw cycles and analyzed for the influence of storage at -80 or 8 °C. We also determined the influence of blood collection and NGS preparations on the miRNA pattern isolated from a single individual, which has been sequenced 10 times. Here, five PAXGene tubes were consecutively collected that have been split in two replicates, representing two experimental batches. All samples were analyzed by Illumina NGS. For each sample, approximately 20 million NGS reads have been generated. Hierarchical clustering and Principal Component Analysis (PCA) showed an influence of the different conditions on the miRNA patterns. The effects of the different conditions on miRNA abundance are, however, smaller than the differences that are due to interindividual variability. We also found evidence for an influence of the NGS measurement on the miRNA pattern. Specifically, hsa-miR-1271-5p and hsa-miR-182-5p showed coefficients of variation above 100% indicating a strong influence of the NGS protocol on the abundance of these miRNAs.
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University , 66123 Saarbrücken, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Backes
authors.affiliation: Institute of Human Genetics, Saarland University , D-66424 Homburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.affiliation: Siemens AG , 80333 Munich, Germany.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Altmann
authors.affiliation: Siemens AG , 80333 Munich, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Wuerstle
authors.affiliation: Internal Medicine II, University Hospital Heidelberg , 69120 Heidelberg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Meder
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University , 66123 Saarbrücken, Germany.
authors.forename: Valentina
authors.initials: V
authors.lastname: Galata
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University , 66123 Saarbrücken, Germany.
authors.forename: Sabine C
authors.initials: SC
authors.lastname: Mueller
authors.affiliation: Siemens AG , 80333 Munich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sickert
authors.affiliation: Siemens AG , 80333 Munich, Germany.
authors.forename: Cord
authors.initials: C
authors.lastname: Stähler
authors.affiliation: Institute of Human Genetics, Saarland University , D-66424 Homburg, Germany.
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University , 66123 Saarbrücken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
mesh_terms: D001793:Blood Preservation; D016000:Cluster Analysis; D019295:Computational Biology; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics*; D025341:Principal Component Analysis; D017423:Sequence Analysis, RNA / Q000639:trends*
chemical_list: D035683:MicroRNAs