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chain reaction (PCR)-based methods. A schematic outline of the assay is shown in Figure 1.
Each Probe Set consists of three groups of oligonucleotides that are complementary to the target mRNA. Capture Extenders (CEs) bind both to the mRNA and to the capture plate, working cooperatively to increase the likelihood that other CEs along the mRNA also bind to the plate. This results in a very stable, specific hybridization event that includes multiple 15-20 bp regions along the length of mRNA. Label Extenders (LEs) bind both to the target mRNA and to the trunk of a branched DNA (bDNA) amplifier. The Blocking Probes (BLs) hybridize to regions not covered by other probe sets. Signal amplification occurs when bDNA amplifiers bind the mRNA via the LEs. Signal amplification is precisely controlled by, and is directly proportional to, the number of LEs in the Probe Set, which is more reproducible than target amplification or PCR. As a result, the final luminescent readout is directly proportional to the quantity of input RNA.
METHODS
Induction of IL-8 gene expression by PMA/LPS in HeLa cells
Approximately 5,000 HeLa cells per well were plated in a 96-well plate in 200 l of DMEM medium (Invitrogen, Carlsbad, CA, USA). At 70% confluence (16-24 hours), the cells were induced with a 100 nM final concentration of Phorbol-12-myrstyl-13-acetate (PMA, Calbiochem, EMD Biosciences, San Diego, CA, USA) in SFM media (GibcoCell Cultures, Invitrogen) for two hours. The siRNAs specific for the IL-8 mRNA (Trilink Biotechnologies, San Diego, CA, USA) were diluted in OptiMEM media (Invitrogen) to a final concentration of 100 nM and added to the Lipofectamine 2000 (Invitrogen) transfection mix. The media bathing the HeLa cells was replaced by the siRNA/Lipofectamine mix to a volume of 175 l. Cells were transfected at 37C for four hours, followed by recovery for either four or eight hours in
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