title: Comparison of whole blood RNA preservation tubes and novel generation RNA extraction kits for analysis of mRNA and MiRNA profiles.
abstract: BACKGROUND
  Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.
  
  
  METHODS
  3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.
  
  
  RESULTS
  RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.
  
  
  CONCLUSIONS
  We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.
authors.affiliation: LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
authors.forename: Madlen
authors.initials: M
authors.lastname: Häntzsch
authors.affiliation: Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Tolios
authors.affiliation: LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; Department of Cardiology, Heart Center, University Leipzig, Leipzig, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Beutner
authors.affiliation: Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Nagel
authors.affiliation: LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Thiery
authors.affiliation: LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Teupser
authors.affiliation: LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Lesca M
authors.initials: LM
authors.lastname: Holdt
mesh_terms: D001793:Blood Preservation / Q000379:methods; D001800:Blood Specimen Collection / Q000295:instrumentation*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000302:isolation & purification*; D009203:Myocardial Infarction / Q000097:blood / Q000235:genetics; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic