Author(s): Yawen Cheng | Wanru Lin | Yun Yen | Linling Chen | Peter Hwu | Qiang Liu | Yafan Wang
Journal: Journal of Biophysical Chemistry
ISSN 2153-036X
Volume: 02;
Issue: 01;
Start page: 70;
Date: 2012;
Original page
Keywords: Electrophoresis | Gene Deletion Detection | Agilent 2100 Bioanalyzer | RD-PCR | NSCLC
ABSTRACT
We applied the Agilent 2100 Bioanalyzer, a microflu- idics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative pro- duct yield ratio (ROY), to detect somatic gene dele- tions in tumor cells. Briefly for such purpose, the Agi- lent 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 Agi- lent 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.
Journal: Journal of Biophysical Chemistry
ISSN 2153-036X
Volume: 02;
Issue: 01;
Start page: 70;
Date: 2012;
Original page
Keywords: Electrophoresis | Gene Deletion Detection | Agilent 2100 Bioanalyzer | RD-PCR | NSCLC
ABSTRACT
We applied the Agilent 2100 Bioanalyzer, a microflu- idics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative pro- duct yield ratio (ROY), to detect somatic gene dele- tions in tumor cells. Briefly for such purpose, the Agi- lent 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 Agi- lent 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.
