TRANSCRIPTOME 2002: From Functional Genomics to Systems
Definition of Sources of Variability and Noise Factors in Affymetrix Expression Profiling
Marina Bakay1, Yi-Wen Chen1, Rehannah Borup1, Po Zhao1, Kanneboyina Nagaraju2, and Eric P Hoffman1, 1Research Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, 2Division of Rheumatology, Johns Hopkins School of Medicine, Baltimore, MD
We present a systematic study of the sources of variability in expression profiling data using 56 RNAs (28 subjects) isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 RNAs from murine cell cultures and tissues (42 Affymetrix U74A v2 arrays). Variables studied included tissue heterogeneity, cRNA probe production, patient diagnosis, and GeneChip hybridizations. We found that the greatest source of variability was often different regions of the same patient muscle biopsy, reflecting variation in cell type content even in a relatively homogeneous tissue such as muscle. Inter-patient variation was also very high (SNP noise). Experimental variation (RNA, cDNA, cRNA, or GeneChip) was minor. Pre-profile mixing of patient cRNA samples effectively normalized both intra- and inter-patient sources of variation, while retaining a high degree of specificity of the individual profiles (86% of statistically significant differences detected by absolute analysis; and 85% by a 4-pairwise comparison survival method).In conclusion, our data using unsupervised cluster analysis and correlation coefficients of 90 RNA samples on 76 oligonucleotide microarrays showed that experimental error was not a significant source of unwanted variability in expression profiling experiments. Major sources of variability were instead from tissue heterogeneity and inter-individual variations.
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