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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 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.
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 100 l fresh complete DMEM. The QuantiGene Lysis Working Reagent was added to each treatment group for an additional 30 minutes. The QuantiGene assay was performed according to manufacturers instructions.
HeLa cells induced with PMA elicit a robust increase in the IL-8 expression. Based on the experimental results (data not shown), we determined that Cyclophilin B (PPIB) demonstrated more stable expression than GAPDH and subsequently was used as the normalization control. Following stimulation and siRNA transfection, the mRNA levels of each gene were measured from four replicate treatments of each experimental condition. The results are shown in Figure 2.
Coefficients of variation (CVs) measured from different treatments ranged from 5 to 15% over a 3-log range of relative luminescence (RLU) values. These CVs allow for the clear distinction of the effects of the siRNA seen at four hours (89% knockdown) from the effects seen at eight hours (94% knockdown). To normalize the results, the IL-8 and Cyclophilin B measurements made from the same replicate (i.e., the same batch of lysed cells) were converted to a ratio (IL-8 RLU to Cyclophilin B RLU). This ratio was calculated for each of the four replicates for each of the four experimental conditions. The results of this analysis are shown in Figure 3, where the error bars indicate one standard deviation. Following normalization, the range of CVs was determined to be 3 to 15%.
In the study described above, the QuantiGene Reagent System demonstrated a high level of precision and reproducibility in experiments that are commonly performed by researchers who utilize RNAi. CVs ranged from 5 to 15%, and from 3 to 15% when normalized to the expression of an endogenous control gene. The results of these experiments demonstrate that the QuantiGene Reagent System is a powerful companion t echnology for experimental programs that employ siRNA technology.
1. Reynolds, A., Leake, D., Boese, Q., et al. Nature Biotechnology, March 22, 3, 326330 (2004).
2. Chiu, Y. and Rana, T.M. Molecular Cell 10, 549561 (2002).
3. Wilber, J.C., and Urdea, M.S. Methods in Molecular Medicine: Hepatitis C, 19, 7178 (1998).