title: Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance.
abstract: BACKGROUND
  Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays.
  
  
  RESULTS
  While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding.
  
  
  CONCLUSIONS
  The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions.
authors.affiliation: Department of Human Genetics, Medical School, Saarland University, Building 60, 66421, Homburg/Saar, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.affiliation: Department of Clinical Bioinformatics, Saarland University, Building E2.1, 66123, Saarbrücken, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Backes
authors.affiliation: Department of Human Genetics, Medical School, Saarland University, Building 60, 66421, Homburg/Saar, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Rheinheimer
authors.affiliation: Department of Clinical Bioinformatics, Saarland University, Building E2.1, 66123, Saarbrücken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
authors.affiliation: Department of Human Genetics, Medical School, Saarland University, Building 60, 66421, Homburg/Saar, Germany.
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D000925:Anticoagulants / Q000737:chemistry*; D015415:Biomarkers / Q000097:blood*; D001793:Blood Preservation / Q000379:methods*; D001800:Blood Specimen Collection; D016000:Cluster Analysis; D004492:Edetic Acid / Q000737:chemistry*; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000187:drug effects / Q000235:genetics; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D018962:Phlebotomy; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA; D013048:Specimen Handling; D013997:Time Factors; D055815:Young Adult
chemical_list: D000925:Anticoagulants; D015415:Biomarkers; C546957:MIRN769 microRNA, human; D035683:MicroRNAs; D004492:Edetic Acid