oct. 23 application note page 1 of 6 RNAi in suspension cells using amaxa s Nucleofector technology together with QIAGEN s HPP Grade sirna duplexes Elke Lorbach*, Dietmar Lenz*, Andrea Klaes*, Katharina Hein*, Bodo Ortmann*, Tatjana Males*, Hanns-Martin Schmidt*, Günter Kraus*, Peter Hahn**, Wolfgang Bielke** *amaxa GmbH, Köln, Germany **Qiagen GmbH, Hilden, Germany Successful sirna gene silencing depends on many different parameters such as mrna turnover, mrna abundance or half-life of the protein. Two important factors that can positively influence your sirna experiment are the design of the sirna oligo and the efficient delivery of the sirna. Here we show, that HPP Grade sirna from QIAGEN works well in combination with amaxa s Nucleofector technology and can be used to efficiently silence genes in various hard-to-transfect suspension cell lines as well as in primary cells. Introduction WTB-13_1.3 RNAi allows targeted knockdown of gene expression by introducing short interfering RNA (sirna), homologous to the mrna of the target gene, into cells. This method of gene silencing has become a valuable tool in functional genomics research, target validation, and gene-specific therapeutics. Successful RNAi experiments are dependent on two major parameters: sirna design and efficient delivery of sirna into cells. QIAGEN and amaxa have joined forces to provide a complete solution for RNA interference (RNAi) that overcomes the problems associated with the key points of sirna design and delivery into mammalian cells. amaxa s R&D team has demonstrated efficient sirna delivery in suspension cells using QIAGEN s HPP Grade sirna and amaxa s Nucleofector technology. In this study, efficient silencing of the housekeeping gene vimentin and the T cell receptor genes, CD2 and CD4, are shown in a variety of suspension cell lines such as JURKAT, U937, THP-1, HL-6 and K562 as well as in non-dividing, unstimulated primary human T cells. amaxa GmbH amaxa Inc. Europe/World USA +49 ()221-99199-4 (24) 632-911 techservice@amaxa.com techservice.us@amaxa.com
oct. 23 application note page 2 of 6 sirna design and synthesis The design and quality of an sirna has a strong influence on the level and specificity of gene silencing. QIAGEN provides an sirna Design Tool that uses state-of-the-art design criteria for easy design of sirna (Figure 1). To further minimize the risk that particular sirna will not result in effective silencing, 4-for-Silencing sirna duplexes are available. These are a set of four HPP (High- Performance Purity) Grade sirna duplexes, designed by QIAGEN scientists, to target the gene of choice. QIAGEN guarantees 7% mrna knockdown with at least one of these sirna duplexes, otherwise four additional sirna duplexes are provided without additional cost. sirna directed against a range of endogenous and reporter genes is available for developing and optimizing sirna experiments. In addition, library sets are available for screening a large number of genes. A regularly updated list can be found at www.qiagen.com/sirna. QIAGEN s HPP Grade sirna is delivered >9% pure eliminating the time and expense needed for HPLC or PAGE purification (Figure 2). sirna design and ordering Intensity 1 9 8 7 6 5 4 3 2 1 5 Mass/Charge 55 6 65 7 75 8 85 Figure 2 High-purity sirna for efficient gene silencing QIAGEN performs MALDI-TOF mass spectrometric analysis on every HPP Grade sirna to confirm its integrity. WTB-13_1.3 Figure 1 Easy sirna design Screenshot of the QIAGEN sirna Design Tool
oct. 23 application note page 3 of 6 sirna delivery with Nucleofector technology JURKAT JURKAT The Nucleofector technology is a non-viral method for transfection of hard-to-transfect cell lines and primary cells. It is based on two components, the Nucleofector Device that delivers unique electrical parameters and the Nucleofector Kits that contain cell-specific and optimized Nucleofector Solutions. Ready-to-go protocols are available for many cell lines and primary cell types. The technology is suitable for the delivery of different substrates such as DNA, RNA or sirna oligos without further optimization of the nucleofection conditions. HL-6 HL-6 Suspension cells are difficult to transfect with conventional transfection methods e.g. lipid-based reagents with both plasmid DNA and sirna oligos. In this study, we show that sirna oligos can be efficiently delivered into many different suspension cell types with the Nucleofector technology and that an effective gene silencing can be achieved as shown by different methods such as flow cytometry, western blot and quantitative RT-PCR. Gene silencing of various genes in suspension cell lines K562 K562 In order to determine the efficiency of nucleofection with sirna duplexes, Rhodamine-labeled sirna [QIAGEN] was transfected into different suspension cell types such as JURKAT, HL-6, K562 and THP-1 cells (Figure 3). Cells were analyzed by fluorescence microscopy and sirna delivery was achieved with up to 99% efficiency. JURKAT, HL-6, K562, THP-1, U937 and primary human T cells were transfected with sirna targeted to the housekeeping gene vimentin [QIAGEN, library sirna; Cat.No. 12255]. 24 and 48 hours after nucleofection, silencing of vimentin THP-1 WTB-13_1.3 THP-1 Figure 3 Up to 99% sirna delivery into suspension cells Different suspension cell lines were nucleofected using the appropriate Nucleofector Kit and Rhodamine-labeled sirna. 3 hours post nucleofection cells were analyzed by light and fluorescence microscopy.
oct. 23 application note page 4 of 6 gene expression was determined by quantitative, real-time RT-PCR (Figure 4). The experiment clearly shows that expression of vimentin mrna was downregulated with silencing effects between 7-9%. Furthermore, it shows that this level of silencing is dependent on the cell type and time of analysis due to different mrna kinetics in different cell types. Knockdown of vimentin was also shown at protein level using western blot. JURKAT cells were transfected with three different concentrations of sirna targeted to the vimentin gene (Figure 5). Already after 24 hours, the amount of vimentin protein was clearly reduced using.7 µg sirna. After 48 hours, the expression was further downregulated and with.7 µg the signal for vimentin was no longer detectable. This shows, that silencing occurs also on protein level and that the silencing effects are dose dependent. 1 vimentin sirna (24h) vimentin sirna (48h) 75 % relative expression 5 25 Human T cells JURKAT THP-1 U937 K562 HL-6 Figure 4 Gene silencing of vimentin in various suspension cell types Six different suspension cell types were transfected with 1.4 µg sirna targeted to vimentin or with 1.4 µg control sirna using the cell-type specific Nucleofector protocols. After 24 and 48 hours total RNA was prepared from cells and used for quantitative real-time RT-PCR analysis. The expression level of vimentin mrna was normalized to the expression of a housekeeping gene and quantified using a standard curve. Vimentin knockdown at protein level in JURKAT cells 24 hours 48 hours kda M No sirna + nucleofection No nucleofection +1.4 µg sirna.14 µg.7 µg 1.4 µg M No sirna + nucleofection.14 µg.7 µg 1.4 µg M 62 Vimentin - 47 Actin - 32 WTB-13_1.3 Figure 5 Vimentin knockdown at protein level in JURKAT cells JURKAT (ATCC) cells were transfected using Nucleofector Kit V, program C-16 and.14 µg,.7 µg, or 1.4 µg sirna targeted to vimentin. Western blot analysis was carried out 24 and 48 hours post nucleofection. Control lanes show samples with sirna added without nucleofection and with nucleofection, but no addition of sirna. The level of actin expression is shown for comparison. M=markers
oct. 23 application note page 5 of 6.14 µg CD4 sirna.35 µg CD4 sirna 18 18 15 15 12 12 9 9 6 3 1 1 1 1 2 1 3 1 4.7 µg CD4 sirna 18 15 12 9 6 3 1 1 1 1 2 1 3 1 4 1.4 µg CD4 sirna 18 15 12 9 6 3 1 1 1 1 2 1 3 1 4 6 3 1 1 1 1 2 1 3 1 4 1. µg CD4 sirna 18 15 12 9 6 3 1 1 1 1 2 1 3 1 4 CD4 sirna Two other suspension cell lines, the promyelocytic suspension cell line HL-6 and the monocytic leukemia cell line THP-1 were used to show gene silencing of the CD4 T cell receptor gene. Both cell types were transfected with sirna targeted to the CD4 gene [QIAGEN, library sirna; Cat.No. 124675]. 48 hours after delivery of sirna, downregulation of CD4 was analyzed by flow cytometry (Figure 6 and 7). In HL-6 cells five different amounts of sirna duplexes were transfected, and down regulation of CD4 expression occurred in a dose-dependent manner. The most pronounced gene silencing effects were achieved with 1.4 µg sirna (Figure 6). In THP-1 cells, 1.4 µg sirna was transfected and effective knockdown of CD4 expression was achieved (Figure 7). Transfection of nonspecific, duplexes did not influence the expression of CD4. WTB-13_1.3 Figure 6 Optimization of sirna amounts in HL-6 cells Five different amounts of sirna duplexes targeting human CD4 were nucleofected into HL-6 cells using Nucleofector Kit V and program T-19. 48 hours after nucleofection, cells were stained with anti-cd4-phycoerythrin (PE) antibody and analyzed by flow cytometry. sirna targeted against vimentin was used as control. 24 18 12 6 1 1 1 1 2 1 3 1 4 CD4 sirna no sirna + nucleofection Figure 7 CD4 gene silencing in THP-1 cells THP-1 cells were transfected using Nucleofector Kit V and program V-1 and 1.4 µg sirna targeted to CD4. 48 hours after nucleofection, cells were stained with anti- antibody and analyzed by flow cytometry. Controls included nucleofection of 1.4 µg and nucleofection without sirna. Successful gene silencing in primary human T cells Efficient delivery of sirna oligonucleotides into primary cells is the key bottleneck before functional genomics can be addressed under in vivo-like conditions such as those found in primary cells. Here we show that primary human T cells can be used for sirna experiments with the Nucleofector technology. Unstimulated human T cells were transfected with sirna targeted to the pan T cell receptor gene, CD2 [sequence published by D. Unutmaz in amaxa appl. note: RNAimediated CD2 suppression in primary human T cells, WTB-11]. 48 hours after nucleofection, the downregulation of CD2 expression could be shown by flow cytometry. Expression of the CD4 receptor was not influenced as demonstrated by control staining with a PE-coupled CD4 antibody
oct. 23 application note page 6 of 6 2 16 12 8 4 A and flow cytometric analysis (Figure 8). This clearly shows that the gene silencing effect achieved was CD2 specific thus proving the Nucleofector technology an efficient tool to transfect sirna duplexes not only into suspension cell lines but also into primary suspension cells. 1 1 1 1 2 1 3 1 4 CD2-FITC 3 B 24 18 12 6 1 1 1 1 2 1 3 1 4 Figure 8 High specificity of CD2 sirna in primary human T cells Primary human T cells were nucleofected with 1.4 µg sirna targeted to CD2 using the Human T Cell Nucleofector Kit. Cells were stained with A anti CD2-FITC antibody or B anti antibody, and analyzed by flow cytometry 44 hours after nucleofection. Controls included nucleofection with 1.4 µg and nucleofection without sirna. CD2 sirna no sirna + nucleofection Conclusions amaxa s and QIAGEN gene silencing services allow: Successful gene silencing by combining knowledge of sirna synthesis, design and delivery Efficient sirna delivery up to 99% transfection efficiency Tested for many cell types validated for JURKAT, THP-1, HL-6, K562, U937, and primary human T cells Comprehensive technical full support from sirna design and synthesis to delivery of sirna support in virtually any cell type For further info on sirna design and synthesis go to www.qiagen.com/sirna. For further info on sirna delivery go to www.amaxa.com/rnai. Trademarks and disclaimers Nucleofector, Nucleofection (amaxa GmbH) QIAGEN (QIAGEN GmbH) sirna technology licensed to QIAGEN is covered by various patent applications, owned by the Massachusetts Institute of Technology, Cambridge, MA, USA and others. WTB-13_1.3 Australia QIAGEN Pty Ltd Orders 3-9489-3666 Fax 3-9489-3888 Technical 1-8-243-66 Canada QIAGEN Inc. Orders 8-572-9613 Fax 8-713-5951 Technical 8-DNA-PREP (8-362-7737) France QIAGEN S.A. Orders 1-6-92-92 Fax 1-6-92-925 Technical 1-6-92-93 Germany QIAGEN GmbH Orders 213-29-12 Fax 213-29-22 Technical 213-29-124 Italy QIAGEN S.p.A. Orders 2-3343411 Fax 2-3343426 Technical 2-3343414 Japan QIAGEN K.K. Telephone 3-5547-811 Fax 3-5547-818 Technical 3-5547-811 Switzerland QIAGEN AG Orders 61-319-3-3 Fax 61-319-3-33 Technical 61-319-3-31 United Kingdom QIAGEN Ltd. Orders 1293-422-911 Fax 1293-422-922 Technical 1293-422-999 USA QIAGEN Inc. Orders 8-426-8157 Fax 8-718-256 Technical 8-DNA-PREP (8-362-7737) amaxa GmbH amaxa Inc. Europe/World USA +49 ()221-99199-4 (24) 632-911 techservice@amaxa.com techservice.us@amaxa.com