title: Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies.
abstract: OBJECTIVE
  Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples.
  
  
  METHODS
  The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7.
  
  
  RESULTS
  Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible.
  
  
  CONCLUSIONS
  This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels.
authors: Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.
authors: Anke
authors: A
authors: Meyer
authors: Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.
authors: Federico
authors: F
authors: Paroni
authors: Leibniz-Institute for Prevention Research and Epidemiology-BIPS, Bremen, Germany.
authors: Kathrin
authors: K
authors: Günther
authors: Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.
authors: Gitanjali
authors: G
authors: Dharmadhikari
authors: Leibniz-Institute for Prevention Research and Epidemiology-BIPS, Bremen, Germany.
authors: Wolfgang
authors: W
authors: Ahrens
authors: Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.
authors: Sørge
authors: S
authors: Kelm
authors: Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.|German Center for Diabetes Research (DZD) project partner, University of Bremen, Bremen, Germany.
authors: Kathrin
authors: K
authors: Maedler
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D051761:Paired Box Transcription Factors / Q000235:genetics*; D010865:Pilot Projects; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic