title: Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays.
abstract: BACKGROUND
  Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted.
  
  
  RESULTS
  14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance.
  
  
  CONCLUSIONS
  Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. gmutter@rics.bwh.harvard.edu
authors.forename: George L
authors.initials: GL
authors.lastname: Mutter
authors.forename: David
authors.initials: D
authors.lastname: Zahrieh
authors.forename: Chunmei
authors.initials: C
authors.lastname: Liu
authors.forename: Donna
authors.initials: D
authors.lastname: Neuberg
authors.forename: David
authors.initials: D
authors.lastname: Finkelstein
authors.forename: Heather E
authors.initials: HE
authors.lastname: Baker
authors.forename: Janet A
authors.initials: JA
authors.lastname: Warrington
mesh_terms: D000704:Analysis of Variance; D005260:Female; D005615:Freezing; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007044:Hysterectomy; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability / Q000235:genetics; D012996:Solutions; D014021:Tissue Preservation / Q000379:methods* / Q000706:statistics & numerical data; D014591:Uterine Diseases / Q000235:genetics
chemical_list: D012996:Solutions; D012313:RNA