Ratio quantification of gene dosage by Agilent 2100 Bioanalyzer for detection of somatic gene deletions

Peter Hwu, Qiang Liu, Yafan Wang, Linling Chen, Yawen Cheng, Wanru Lin, Yun Yen

Department of Molecular Pharmacology, City of Hope Comprehensive Cancer Center, Duarte, USA.
Institute of Medicine, Chung Shan Medical University, Taichung, Chinese Taipei.
Translational Research Core Laboratory, City of Hope Comprehensive Cancer Center and Beckman Research Institute, Duarte, USA.

DOI: 10.4236/abc.2012.21009                

Abstract

We applied the Agilent 2100 Bioanalyzer, a microfluidics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative product yield ratio (ROY), to detect somatic gene deletions in tumor cells. Briefly for such purpose, the Agilent 2100 Bioanalyzer quantified the ROY of a target gene to an endogenous internal control, both of which were initially co-amplified by Robust-Dosage PCR (RD- PCR). Herein, we extensively validated this approach. We first tested the effect of well positions on the Agilent DNA chip. We found that no matter which wells the samples were loaded in, the ROY was consistent with coefficient of variation (CV) < 2%. Then we tested the effect of product concentrations that varied 8-fold, and the ROY was also consistent with CV < 3.5%. Furthermore, we applied this approach to identify six somatic KRAS deletions in non-small cell lung cancer patients, confirming our previous findings. Thus, the Agilent 2100 Bioanalyzer is simple, accurate, quick, and ultimately able to replace conventional gel electrophoresis for the detection of somatic gene deletions.

Keywords

Electrophoresis; Gene Deletion Detection; Agilent 2100 Bioanalyzer; RD-PCR; NSCLC

Conflicts of Interest

The authors declare no conflicts of interest.

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