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RNAi optimization and control experiments Christian Korfhage, Peter Hahn, Andreas Meier, Gesa Niemann, Wolfgang Bielke, and Dirk Löffert QIAGEN GmbH, Hilden, Germany Important factors in the success of RNAi studies are optimization of transfection and performance of suitable control experiments that prevent artifactual effects being wrongly interpreted. In this study, we demonstrate that the RNAi Human/Mouse Control Kit, used with QuantiTect ® Gene Expression Assays for downstream analysis, allows convenient siRNA transfection optimization and control experiments in both human and mouse cells. Alexa Fluor Label Allows Easy Monitoring of Transfection Figure 1 HeLa S3 cells were transfected with Alexa Fluor 488 labeled, non-silencing control siRNA using RNAiFect Reagent. Transfection efficiency was determined using fluorescence microscopy 24 hours after transfection. QuantiTect Gene Expression Assays Provide Highly Sensitive Quantitative RT-PCR Results 6 5 4 3 2 1 0 HeLa S3 NIH/3T3 1 10 ng total RNA 100 Figure 2 Total RNA from untransfected HeLa S3 and NIH/3T3 cells was used in quantitative RT-PCR analysis with the human and mouse MAPK1 Gene Expression Assays. Standard curves show CT values obtained from RNA dilutions normalized against CT values obtained using 100 ng total RNA. Convenient optimization of transfection and positivecontrol experiments The RNAi Human/Mouse Control Kit includes a non-silencing Alexa Fluor ® 488 labeled siRNA for transfection optimization. The long duration of Alexa Fluor fluorescence, and its intensity in cells transfected with low siRNA concentrations, mean that it is very useful for optimization of RNAi experiments. A validated siRNA targeted against a sequence common to both the human and mouse MAPK1 gene is also provided in the kit. The protein kinase MAPK1 (also called Erk2 and MAPK2) is highly expressed in a wide variety of human and mouse cell types. The MAPK1 siRNA almost eliminates MAPK1 expression in human and mouse cells making it a highly suitable positive control. RNAiFect Transfection Reagent provides high siRNA transfection efficiency and is also included in the control kit. Accurate analysis of knockdown efficiency Quantitative, real-time RT-PCR is frequently used to analyze the results of gene silencing experiments. QuantiTect Gene Expression Assays are an expanding range of functionally validated primer–probe sets that are ready to use for optimal results in quantitative, real-time RT-PCR on the majority of real-time cyclers. The assays comprise two gene-specific primers and a dual-labeled QuantiProbe in a 10x Assay Mix. Optimal results are guaranteed when QuantiTect Gene Expression Assays are used in combination with QuantiTect Probe Kits. All QuantiTect Assays can be used for one-step RT-PCR using the QuantiTect Probe RT-PCR Kit, or two-step RT-PCR using the QuantiTect Probe PCR Kit. The assays and kits have been optimized and validated to ensure high PCR efficiency and accurate quantification of as few as 10 copies of template. Primers are designed to cross exon-exon boundaries, so that only RNA sequences are amplified and detected. Materials and methods Non-silencing and positive-control siRNA duplexes from the RNAi Human/Mouse Control Kit were transfected into HeLa S3, HEK 293, or NIH/3T3 cells. Transfection efficiency of the Alexa Fluor labeled 66 www.qiagen.com QIAGEN News 2004 Issue 3
non-silencing siRNA was examined by fluorescence microscopy. RNA was purified using the RNeasy ® Mini Kit. MAPK1 knockdown using the positive control siRNA was determined using MAPK1-specific QuantiTect Gene Expression Assays. The QuantiTect Hs_MAPK1 Assay and the QuantiTect Mm_Mapk1 Assay, for human and mouse respectively, were used with the QuantiTect Probe RT-PCR Kit for one-step RT-PCR or with Omniscript ® Reverse Transcriptase and the QuantiTect Probe PCR Kit for two-step RT-PCR. Results Transfection of Alexa Fluor 488 labeled, non-silencing siRNA allowed determination of transfection efficiency by fluorescence microscopy (Figure 1). The transfection efficiency in HeLa S3 cells was close to 100%. The Alexa Fluor 488 fluorophore is brighter and more photostable than other fluorescent labels. It is tolerant of pH changes within a wide range, making it very stable in living cells. The QuantiTect Assays for MAPK1 were used to generate standard curves using dilutions of RNA purified from untransfected HeLa S3 and NIH/3T3 cells respectively (Figure 2). Linear standard curves show that QuantiTect Gene Expression Assays are highly sensitive and accurate, as ∆CT values correlate closely to RNA amount used. Human and mouse cell lines transfected with MAPK1 siRNA were analyzed for gene silencing efficiency using the QuantiTect Assays for MAPK1. One-step or two-step RT-PCR was carried out on either the ABI GeneAmp ® 5700 or ABI PRISM ® 7700 real-time cycler (Figure 3). High levels of MAPK1 mRNA knockdown were achieved, showing that the MAPK1 siRNA is a highly suitable positive control for gene silencing in both human and mouse cells. QuantiTect Gene Expression Assays provided accurate results for both one-step and two-step RT-PCR, and with both real-time cyclers. This demonstrates the flexibility of QuantiTect Gene Expression Assays for use with different real-time cyclers and reaction setups. Conclusions ■ The RNAi Human/Mouse Control Kit and QuantiTect Gene Expression Assays for human and mouse MAPK1 can be used as integrated tools for gene silencing and downstream RT-PCR analysis. These tools can be routinely used as controls for both the transfection and downstream analysis stages of RNAi experiments. ■ Standard curves generated using RNA dilutions show that QuantiTect Gene Expression Assays are highly accurate and sensitive. ■ QuantiTect Gene Expression Assays provided optimal results for both one- and two-step RT-PCR on different real-time cyclers. Quantitative Real-Time RT-PCR Shows MAPK1 Knockdown QIAGEN News 2004 Issue 3 www.qiagen.com 67 A Relative MAPK1 mRNA expression (%) B Relative MAPK1 mRNA expression (%) 120 100 80 60 40 20 0 120 100 80 60 40 20 0 HeLa S3 Control One-step, ABI PRISM 7700 Two-step, ABI PRISM 7700 Two-step, GeneAmp 5700 NIH/3T3 HEK 293 Figure 3 A Human cell lines HeLa S3 and HEK 293 and B mouse cell line NIH/3T3 cells were transfected with nonsilencing control siRNA or siRNA targeted against MAPK1 mRNA using RNAiFect Reagent. After 48 hours, one-step or two-step quantitative, real-time RT-PCR analysis was carried out using the appropriate QuantiTect Gene Expression Assay and the indicated real-time cycler.
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