Open Access Highly Accessed Research article

A sequence-based approach to identify reference genes for gene expression analysis

Raj Chari1*, Kim M Lonergan1, Larissa A Pikor1, Bradley P Coe1, Chang Qi Zhu2, Timothy HW Chan13, Calum E MacAulay1, Ming-Sound Tsao2, Stephen Lam1, Raymond T Ng13 and Wan L Lam1

Author Affiliations

1 Department of Integrative Oncology, British Columbia Cancer Agency Research Centre, Vancouver, BC, Canada

2 Ontario Cancer Institute/Princess Margaret Hospital, Toronto, ON, Canada

3 Department of Computer Science, University of British Columbia, Vancouver, BC, Canada

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BMC Medical Genomics 2010, 3:32  doi:10.1186/1755-8794-3-32

Published: 3 August 2010

Additional files

Additional file 1:

Summary of lung SAGE libraries. Raw tag count summary for lung SAGE libraries used in analysis.

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Additional file 2:

Summary of breast SAGE libraries. Raw tag count summary for breast SAGE libraries used in analysis.

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Additional file 3:

Summary of brain SAGE libraries. Raw tag count summary for brain SAGE libraries used in analysis.

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Additional file 4:

Taqman probe IDs of genes assessed by quantitative RT-PCR. Applied Biosystems Taqman probe IDs for genes assessed by qRT-PCR.

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Additional file 5:

Probes used for NEPS genes represented on the Affymetrix U133A microarray. Probes for NEPS genes represented on the Affymetrix U133A microarray.

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Additional file 6:

Probes used for NEPS genes represented on the Agilent microarray. Probes used for NEPS genes represented on the Agilent microarray.

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Additional file 7:

Permutation scores of previously identified and NEPS identified reference genes. Data used in scatter plot shown in Figure 1.

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Additional file 8:

Ingenuity Pathway Analysis using genes from the analyses of a NEPS-normalized and unnormalized dataset by Landi et al. Ingenuity Pathway Analysis using genes from the analyses of a NEPS-normalized and unnormalized dataset by Landi et al.

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Additional file 9:

SAM and pathway analysis of an Agilent lung cancer microarray dataset normalized with and without lung NEPS genes. SAM and pathway analysis of a dataset normalized with and without lung NEPS genes. (A) Number of probes identified as differentially over and underexpressed between cancer and normal using SAM on the dataset with and without NEPS normalization. Venn diagram illustrates the overlap in the genes identified as well as those which are different between the two analyses. (B) Canonical pathway analysis using Ingenuity Pathway Analysis. Dark blue bars represent the results from the dataset normalized with NEPS and median normalization and light blue bars represent the results from using median normalization alone. While similar pathways are statistically significant, each pathway is slightly different in the degree of statistical significance. Such differences illustrate the impact of reference gene selection and normalization on differential gene expression analysis.

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Additional file 10:

Ingenuity Pathway Analysis using genes from the analyses of a NEPS-normalized and unnormalized dataset by Boelens et al. Ingenuity Pathway Analysis using genes from the analyses of a NEPS-normalized and unnormalized dataset by Boelens et al.

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