title: RNA extraction from healthy and failing human myocardium: a comparative evaluation.
abstract: BACKGROUND
  Isolation of high-quality RNA from tissue is mandatory for producing reliable data for downstream applications. In heart tissue, the relative strengths and weaknesses of different approaches to isolate total RNA are unknown. The objective of this study was to compare different RNA isolation methods in healthy and diseased human myocardium.
  
  
  METHODS
  Frozen left ventricular myocardium was obtained from individuals with heart failure and individuals who died from non-cardiac causes with normal heart function (control). Three extraction methods, including guanidine isothiocyanate (TRIzol), silica-gel column (RNeasy), and the combination method (TRIzol/RNeasy), were assessed for their effect on the yield, integrity, and gene expression levels of RNA using quantitative real-time PCR.
  
  
  RESULTS
  In the control group (n=5), the highest RNA yield per tissue mass was obtained with TRIzol, and a significantly higher RNA integrity was obtained from the RNeasy method. The quantification cycle (Cq) values for both the reference gene GAPDH and two target genes were lower with TRIzol. Normalization by GAPDH showed the highest gene expression levels with RNeasy. Similar patterns were observed in the heart failure group (n=5), suggesting assays were not negatively impacted by myocardial disease processes.
  
  
  CONCLUSIONS
  In both healthy and diseased heart tissue, the TRIzol method provides the highest RNA yield, while the RNeasy method shows superior RNA integrity, demonstrating comparable RNA quality in studies examining myocardial disease. A balanced approach to RNA quality is necessary for the successful downstream applications of RNA.
authors.affiliation: 1 Division of Cardiology, The Heart Institute, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio.
authors.forename: Yaejee H
authors.initials: YH
authors.lastname: Hong
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Martin
authors.forename: Jason M
authors.initials: JM
authors.lastname: Mulvaney
authors.forename: Michael S
authors.initials: MS
authors.lastname: Burhans
authors.forename: Burns C
authors.initials: BC
authors.lastname: Blaxall
authors.forename: Robert B
authors.initials: RB
authors.lastname: Hinton
mesh_terms: D015925:Cryopreservation; D020869:Gene Expression Profiling; D006146:Guanidines / Q000737:chemistry; D006333:Heart Failure / Q000235:genetics / Q000473:pathology*; D006352:Heart Ventricles / Q000737:chemistry* / Q000473:pathology; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D008297:Male; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D020871:RNA Stability; D058428:Silica Gel / Q000737:chemistry
chemical_list: D006146:Guanidines; D017879:Isothiocyanates; C054435:guanidine isothiocyanate; D058428:Silica Gel; D012313:RNA