By Genospectra, Inc., 6519 Dumbarton Circle, Fremont, CA 94555.
A critical step in siRNA-mediated gene expression knockdown studies is the validation of expression inhibition. The QuantiGene Reagent System and the Molecular Devices LMax II384 microplate luminometer were used to measure IL-8 mRNA in PMA-induced HeLa cells, following siRNA treatment. Quantitative assessment of the knockdown discerned differences of less than 10% in mRNA expression levels.
There are many sources of experimental variation that make accurate quantification of mRNA difficult when standard methods are employed. Potency and stability of siRNA coupled with variation in transfection efficiencies lead to varying effectiveness of knocking down of the target genes mRNA(1, 2) and, thus, to variability observed in RNA interference (RNAi) experiments.2 Compounding these factors are variability and inaccuracy arising from differences in sample loss during the RNA purification steps, as well as sequence-specific and random biases that occur during enzymatic amplification processes. This application note presents the QuantiGene Reagent System as an alternative method for direct RNA quantification directly from cell lysates. The siRNA-mediated knockdown of IL-8 gene expression in induced HeLa cells is employed as a model system.
QuantiGene assay principle
Bypassing many of these variability issues, the QuantiGene assay allows quantification of RNA levels directly from fresh or fixed cell lysates or tissue homogenates. This technology, based on branched DNA, allows for amplification of the signal, rather than the target, following the cooperative hybridization between the target mRNA and a gene-specific Probe Set.3 Signal amplification is precisely controlled by the Probe Set design and, therefore, is more reproducible than polymerase