title: Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.
abstract: BACKGROUND
  Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study.
  
  
  RESULTS
  Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this "gold-standard" comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues.
  
  
  CONCLUSIONS
  Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.
authors.forename: Arianne C
authors.initials: AC
authors.lastname: Richard
authors.forename: Paul A
authors.initials: PA
authors.lastname: Lyons
authors.forename: James E
authors.initials: JE
authors.lastname: Peters
authors.forename: Daniele
authors.initials: D
authors.lastname: Biasci
authors.forename: Shaun M
authors.initials: SM
authors.lastname: Flint
authors.forename: James C
authors.initials: JC
authors.lastname: Lee
authors.forename: Eoin F
authors.initials: EF
authors.lastname: McKinney
authors.forename: Richard M
authors.initials: RM
authors.lastname: Siegel
authors.affiliation: Cambridge Institute for Medical Research and Department of Medicine, University of Cambridge, Cambridge, UK. kgcs2@cam.ac.uk.
authors.forename: Kenneth G C
authors.initials: KG
authors.lastname: Smith
mesh_terms: D056648:Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis / Q000235:genetics / Q000276:immunology; D016022:Case-Control Studies; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000235:genetics / Q000276:immunology; D007962:Leukocytes / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009928:Organ Specificity; D012313:RNA / Q000235:genetics*; D013223:Statistics as Topic / Q000379:methods*
chemical_list: D012313:RNA
references.citation: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references.pmid: 12184808
references.citation: Nucleic Acids Res. 2008 Oct;36(17):e108
references.pmid: 18676452
references.citation: Nat Methods. 2007 Nov;4(11):911-3
references.pmid: 17906632
references.citation: BMC Biotechnol. 2011 May 09;11:46
references.pmid: 21549012
references.citation: PLoS One. 2011 Feb 28;6(2):e17238
references.pmid: 21386892
references.citation: Biotechniques. 2005 Jul;39(1):75-85
references.pmid: 16060372
references.citation: Bioinformatics. 2004 Feb 12;20(3):323-31
references.pmid: 14960458
references.citation: BMC Bioinformatics. 2009 Jan 08;10:11
references.pmid: 19133141
references.citation: Nat Biotechnol. 2008 Mar;26(3):317-25
references.pmid: 18278033
references.citation: Nat Rev Genet. 2010 Oct;11(10):733-9
references.pmid: 20838408
references.citation: J Biomol Tech. 2004 Sep;15(3):155-66
references.pmid: 15331581
references.citation: Nat Med. 2010 May;16(5):586-91, 1p following 591
references.pmid: 20400961
references.citation: Bioinformatics. 2006 Apr 1;22(7):789-94
references.pmid: 16410320
references.citation: Nat Methods. 2005 May;2(5):337-44
references.pmid: 15846360
references.citation: Biostatistics. 2007 Jan;8(1):118-27
references.pmid: 16632515
references.citation: J Clin Invest. 2011 Oct;121(10):4170-9
references.pmid: 21946256
references.citation: Proc Natl Acad Sci U S A. 2010 May 25;107(21):9546-51
references.pmid: 20460310
references.citation: BMC Bioinformatics. 2006 Mar 15;7:137
references.pmid: 16539732
references.citation: Nucleic Acids Res. 2003 Feb 15;31(4):e15
references.pmid: 12582260
references.citation: BMC Bioinformatics. 2008 Jun 17;9:284
references.pmid: 18559105
references.citation: Bioinformatics. 2009 Feb 1;25(3):415-6
references.pmid: 19106121
references.citation: BMC Bioinformatics. 2010 Feb 24;11:104
references.pmid: 20181266
references.citation: Bioinformatics. 2010 Oct 1;26(19):2363-7
references.pmid: 20688976
references.citation: RNA. 2013 Jan;19(1):51-62
references.pmid: 23169800
references.citation: Brief Bioinform. 2013 Jul;14(4):469-90
references.pmid: 22851511
references.citation: Nat Rev Genet. 2006 Jan;7(1):55-65
references.pmid: 16369572
references.citation: BMC Genomics. 2007 Mar 05;8:64
references.pmid: 17338817
references.citation: Genome Biol. 2002 Jun 14;3(7):RESEARCH0033
references.pmid: 12184807
references.citation: BMC Bioinformatics. 2006 Aug 30;7:395
references.pmid: 16942624
references.citation: Nat Biotechnol. 2006 Sep;24(9):1115-22
references.pmid: 16964225
references.citation: Nucleic Acids Res. 2002 May 15;30(10):e48
references.pmid: 12000853
references.citation: Nat Biotechnol. 2006 Sep;24(9):1123-31
references.pmid: 16964226
references.citation: Anal Biochem. 2006 May 1;352(1):50-60
references.pmid: 16545767
references.citation: Methods Mol Biol. 2011;671:295-320
references.pmid: 20967638
references.citation: Bioinformatics. 2003 Jan 22;19(2):185-93
references.pmid: 12538238
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D1011-5
references.pmid: 21177656
references.citation: Pharmacogenomics J. 2010 Aug;10(4):278-91
references.pmid: 20676067