Methodology article

Universal Reference RNA as a standard for microarray experiments

Natalia Novoradovskaya¹ , Michael L Whitfield^2,5 , Lee S Basehore¹ , Alexey Novoradovsky¹ , Robert Pesich² , Jerry Usary³ , Mehmet Karaca³ , Winston K Wong¹ , Olga Aprelikova⁴ , Michael Fero² , Charles M Perou³ , David Botstein^2,6 and Jeff Braman¹

¹Stratagene, 11011 N. Torrey Pines Road, La Jolla, CA 92037, USA

²Department of Genetics, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA

³Departments of Genetics and Department of Pathology and Laboratory Science, Lineberger Comprehensive Cancer Center, CB# 7295, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

⁴National Cancer Institute, NIH, Bethesda, MD 20892, USA

⁵Department of Genetics, Dartmouth Medical School, 7400 Remsen, Hanover, NH 03755, USA

⁶Lewis-Sigler Institute for Integrative Genomics 140 Carl Icahn Laboratory, Princeton University Washington Road, Princeton, NJ 08544, USA

author email corresponding author email

BMC Genomics 2004, 5:20doi:10.1186/1471-2164-5-20

Published:	9 March 2004

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).

Conclusion

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.