Zhijian J. Chen

Association of ABCA2 expression with determinants of Alzheimer's disease

With the use of a novel method for detecting differential gene expression, alterations in functional gene clusters related to transport or oxidative stress response and β‐amyloid (Aβ) peptide metabolism were identified in a HEK293 cell line engineered to overexpress the human ATP binding cassette transporter ABCA2. These included fatty acid binding protein, phospholipid binding protein, phospholipid synthesis protein, transporter cofactors, seladin‐1, Aβ precursor protein (APP), vimentin, and low‐density lipoprotein receptor‐related protein. ABCA2 was highly expressed in neuroblastoma cells and colocalized with Aβ and APP. Additionally, increased APP protein levels were detected within ABCA2/APP double‐transfected cells, and increased Aβ was detected in the media of ABCA2‐transfected cells relative to controls. The transporter was abundant in the temporal and frontal regions of both normal and Alzheimers disease (AD) brain but was detected at lower concentrations in the parietal, occipital, and cerebellar regions. The ABCA2 transfected cell line expressed resistance to a free radical initiator, confirming involvement in protection against reactive oxygen species and suggesting a further possible link to AD.

Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE)

BackgroundThe ADGE technique is a method designed to magnify the ratios of gene expression before detection. It improves the detection sensitivity to small change of gene expression and requires small amount of starting material. However, the throughput of ADGE is low. We integrated ADGE with DNA microarray (ADGE microarray) and compared it with regular microarray.ResultsWhen ADGE was integrated with DNA microarray, a quantitative relationship of a power function between detected and input ratios was found. Because of ratio magnification, ADGE microarray was better able to detect small changes in gene expression in a drug resistant model cell line system. The PCR amplification of templates and efficient labeling reduced the requirement of starting material to as little as 125 ng of total RNA for one slide hybridization and enhanced the signal intensity. Integration of ratio magnification, template amplification and efficient labeling in ADGE microarray reduced artifacts in microarray data and improved detection fidelity. The results of ADGE microarray were less variable and more reproducible than those of regular microarray. A gene expression profile generated with ADGE microarray characterized the drug resistant phenotype, particularly with reference to glutathione, proliferation and kinase pathways.ConclusionADGE microarray magnified the ratios of differential gene expression in a power function, improved the detection sensitivity and fidelity and reduced the requirement for starting material while maintaining high throughput. ADGE microarray generated a more informative expression pattern than regular microarray.

Amplified differential gene expression microarray.

Amplified Differential Gene Expression (ADGE) and DNA microarray provides a new concept that the ratios of differentially expressed genes are magnified prior to detecting them. The ratio magnification is achieved with the integration of DNA reassociation and polymerase chain reaction (PCR) amplification and ensured with the design of the adapters and primers. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. The combination of ADGE and DNA microarray provides a mutual complement of their strengths: the magnification of ratios of differential gene expression improves the detection sensitivity; the PCR amplification and efficient labeling enhance the signal intensity and reduce the requirement for large amounts of starting material; and the high throughput for DNA microarray is maintained.

Integration of Amplified Differential Gene Expression (ADGE) and DNA Microarray

Amplified Differential Gene Expression (ADGE) provides a new concept that the ratios of differentially expressed genes are magnified before detection in order to improve both sensitivity and accuracy. This technology is now implemented with integration of DNA reassociation and PCR. The ADGE technique can be used either as a stand‐alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. These two techniques are mutually synergistic: the quadratic magnification of ratios of differential gene expression achieved by ADGE improves the detection sensitivity and accuracy; the PCR amplification of templates enhances the signal intensity and reduces the requirement for large amounts of starting material; the high throughput for DNA microarray is maintained.