Open Access Highly Accessed Methodology article

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

Kim M Clark-Langone1*, Jenny Y Wu1, Chithra Sangli1, Angela Chen1, James L Snable1, Anhthu Nguyen1, James R Hackett1, Joffre Baker1, Greg Yothers2, Chungyeul Kim2 and Maureen T Cronin1

Author Affiliations

1 Genomic Health, Inc. Redwood City, CA, USA

2 National Surgical Adjuvant Breast and Bowel Project (NSABP), Pittsburgh, PA, USA

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BMC Genomics 2007, 8:279  doi:10.1186/1471-2164-8-279

Published: 15 August 2007

Additional files

Additional file 1:

Random hexamer versus gene specific priming using highly degraded RNA as a template. The 21 genes from the OncotypeDX Breast Cancer assay were assayed using highly fragmented RNA as template. Random hexamer priming (RH) or gene specific pool (GSP) priming in the reverse transcription reaction were compared. Each gene was run in triplicate wells and the data shown represents the average and standard deviation. The increased specificity of GSP priming allows even the most highly degraded sample to be amplified with robust signal.

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Additional file 2:

Random hexamer versus gene specific priming using in tact RNA as a template. The 21 genes from the OncotypeDX Breast Cancer assay were assayed using Commercially available, in tact RNA as template. Random hexamer priming (RH) or gene specific pool (GSP) priming in the reverse transcription reaction were compared. Each gene was run in triplicate wells and the data shown represents the average and standard deviation. There is only a modest benefit from using GSP priming versus random priming when the template is of high quality.

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Additional file 3:

Gene list and associated primer and probe oligo sequences. Listed are the Official Symbols of all genes analyzed in the study. The forward, reverse and probe oligo sequences are also shown. Probes were dual labelled 5'FAM as a reporter and either 3'BHQ-1 or 3'BHQ-2 as a quencher.

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