Diane D. Ilsley

A microfluidic system for high-speed reproducible DNA sizing and quantitation.

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer‐controlled instrumentation for performing electrophoretic separations and fluorescence detection of double‐stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.

Different delivery methods-different expression profiles.

Recent publications by several groups of researchers have suggested that small interfering RNAs (siRNA) delivered by lipid-mediated transfection induce both sequence-specific effects1 and broad, class-specific changes in gene expression1, 2, 3, 4. These findings challenge convictions previously held in the RNA interference (RNAi) community that assert virtual sequence specificity of siRNA knockdown, and they bring into question the value of this methodology as a research and therapeutic tool.

Gene profiling study of G3139- and Bcl-2-targeting siRNAs identifies a unique G3139 molecular signature

G3139 is a phosphorothioate oligodeoxyribonucleotide that is targeted to the initiation codon region of the Bcl-2 mRNA, which downregulates Bcl-2 protein and mRNA expression via an antisense mechanism. In previous work, we have demonstrated that the phenotype observed in several prostate and melanoma cell lines after treatment with G3139 appears to be Bcl-2 independent. In contrast, downregulation of Bcl-2 expression by a small interfering RNA (siRNA) produced little or no phenotype change. In the present work, we performed an Agilent oligonucleotide microarray assay on mRNA isolated from PC3 prostate cancer cells that were treated with two different oligonucleotide gene-silencing reagents. G3139 and a Bcl-2-targeting siRNA both downregulate Bcl-2 expression, but via different mechanisms. A side-by-side comparative analysis showed that the expression profile generated by these molecules differs substantially. The study revealed upregulation of the expression of stress-inducible genes in PC3 cells at 1 and 3 days after a 5-h transfection with G3139 complexed with Lipofectamine 2000. In contrast, only a very diminished stress response was seen 1 and 3 days after a 24-h transfection of siRNA/Lipofectamine 2000 complexes. These results highlight the profound differences in off-target effects in cells treated with the phosphorothioate oligonucleotide G3139 and with an siRNA targeted to the same gene, and provide further evidence that the mechanism of action of G3139 is not related to Bcl-2 silencing.