title: Universal Reference RNA as a standard for microarray experiments.
abstract: BACKGROUND
  Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment.
  
  
  RESULTS
  Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97).
  
  
  CONCLUSIONS
  Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.
authors.affiliation: Stratagene, 11011 N, Torrey Pines Road, La Jolla, CA 92037, USA. nnovo@stratagene.com
authors.forename: Natalia
authors.initials: N
authors.lastname: Novoradovskaya
authors.forename: Michael L
authors.initials: ML
authors.lastname: Whitfield
authors.forename: Lee S
authors.initials: LS
authors.lastname: Basehore
authors.forename: Alexey
authors.initials: A
authors.lastname: Novoradovsky
authors.forename: Robert
authors.initials: R
authors.lastname: Pesich
authors.forename: Jerry
authors.initials: J
authors.lastname: Usary
authors.forename: Mehmet
authors.initials: M
authors.lastname: Karaca
authors.forename: Winston K
authors.initials: WK
authors.lastname: Wong
authors.forename: Olga
authors.initials: O
authors.lastname: Aprelikova
authors.forename: Michael
authors.initials: M
authors.lastname: Fero
authors.forename: Charles M
authors.initials: CM
authors.lastname: Perou
authors.forename: David
authors.initials: D
authors.lastname: Botstein
authors.forename: Jeff
authors.initials: J
authors.lastname: Braman
mesh_terms: D000818:Animals; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D012313:RNA / Q000235:genetics* / Q000378:metabolism / Q000592:standards; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D012313:RNA