title: Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design.
abstract: BACKGROUND
  Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background.
  
  
  RESULTS
  ERCC pools were analyzed on four different microarray platforms: Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms: the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate.
  
  
  CONCLUSIONS
  The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics: linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies.
authors.affiliation: Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, 443 Via Ortega, Stanford, CA, 94305, USA. p.scott.pine@nist.gov.
authors.forename: P Scott
authors.initials: PS
authors.lastname: Pine
authors.affiliation: Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, 443 Via Ortega, Stanford, CA, 94305, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Munro
authors.affiliation: Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, 443 Via Ortega, Stanford, CA, 94305, USA.
authors.forename: Jerod R
authors.initials: JR
authors.lastname: Parsons
authors.affiliation: Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, 443 Via Ortega, Stanford, CA, 94305, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: McDaniel
authors.affiliation: Genomics Research and Development, Agilent Technologies, Santa Clara, CA, 95051, USA.
authors.forename: Anne Bergstrom
authors.initials: AB
authors.lastname: Lucas
authors.affiliation: Illumina, Inc., San Diego, CA, 92122, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Lozach
authors.affiliation: National Institute of Allergy and Infectious Diseases, Bethesda, MD, 20892, USA.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Myers
authors.affiliation: National Institute of Allergy and Infectious Diseases, Bethesda, MD, 20892, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Su
authors.affiliation: AIBioTech, Inc., Richmond, VA, 23235, USA.|Present Address: GENETWORx, LLC., Glen Allen, VA, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Jacobs-Helber
authors.affiliation: Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, 443 Via Ortega, Stanford, CA, 94305, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Salit
mesh_terms: D000465:Algorithms; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D012313:RNA / Q000235:genetics* / Q000592:standards*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000592:standards*
chemical_list: D012313:RNA
keywords: ERCC; Gene expression; Microarray; RNA controls; RNA sequencing; RNA-Seq; Spike-in controls