Optimized Protocol sirna Test Kit for Cell Lines and Adherent Primary Cells

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1 page 1 of 8 sirna Test Kit for Cell Lines and Adherent Primary Cells Kit principle Co-transfection of pmaxgfp, encoding the green fluorescent protein (GFP) from Pontellina p. with an sirna directed against maxgfp in your cells of interest. Successful gene silencing is monitored as decrease of green fluorescence compared to control sample using fluorescence microscopy. Note This kit has been designed to enable an easy set-up of sirna transfections in your cells of interest. In subsequent sirna experiments, it can be used as a positive control. The kit is not suitable for use with primary blood cells for which we recommend a separate protocol. Example of sirna mediated gene silencing of maxgfp pmaxgfp 0.5 µg 1 µg 2 µg no sirna Data to come +1.5 µg sirna NIH3T3 cells (ATTC) were nucleofected with 0.5, 1 or 2 µg of pmaxgfp only or 1.5 µg sirna directed against max GFP, in additon. Gene silencing of maxgfp expression was monitored by fluorescence microscopy, 24 hours post-nucleofection. Chapter Contents 1 Procedure outline & important advice 2 Product description 3 Additional information 3.1 General remarks 3.2 Initial experimental set-up 3.3 Cell culture 3.4 Information about pmaxgfp 4 Protocol for suspension cells 5 Protocol for adherent cells

2 page 2 of 8 A simple strategy to set up sirna in your cells of interest primary cells cell lines Optimized Protocol available / yes Optimized Protocol available / no Choose the appropriate Nucleofector Kit and Optimized Protocol for cell type of interest. Use Cell Line Optimization Nucleofector Kit. Optimize nucleofection conditions with pmaxgfp plasmid according to the optimization strategy: Solution R T V program 1 A-23 A-23 A-23 program 2 A-27 A-27 A-27 program 3 T-20 T-20 T-20 program 4 T-27 T-27 T-27 program 5 T-16 T-16 T-16 program 6 T-01 T-01 T-01 program 7 G-16 G-16 G-16 program 8 O-17 O-17 O-17 + Use recommended Nucleofector TM program for sirna transfections. Use optimized nucleofection conditions (i.e. Nucleofector TM Solution and program) to establish sirna experiments. maxgfp (3486 bp) Use sirna Test Kit to establish sirna in your cells of interest. Use sirna Test Kit to establish sirna in your cells of interest. Perform gene silencing with your gene of interest and include sirna Test Kit as a positive control. Perform gene silencing with your gene of interest and include sirna Test Kit as a positive control.

3 page 3 of 8 2 Product description Cat. No. VSC-1001 Kit components 75 µg sirna against maxgfp # 9 ml sirna Suspension Buffer* 100 µg pmaxgfp (0.5 µg/µl in 10mM Tris ph 8.0) Size reactions Storage and stability Store all reagents at -20 C. Repeated freeze-thaw cycles will not interfere with the sirna sample (dissolved or lyophilized) as long as RNAse-free conditions are strictly maintained. After thawing, spin the tube briefly, to bring contents to the bottom of the tube. The expiry date is printed on the Kit Label. 3 3 Protocol 3.1 General remarks Using the Nucleofector TM technology, sirna and DNA are transfected using the same conditions, i.e. Nucleofector TM Solution and program. If the optimal nucleofection conditions are not known, e.g. for a specific cell line, we recommend using our Cell Line Optimization Kit (Cat.No. VCO-1001) to determine those. Generally, it is not advisable to optimize nucleofection conditions using fluorescently labeled sirna duplexes. Unless analyzed by confocal fluorescence microscopy, exact transfection efficiencies are difficult to determine. The easiest way to monitor gene silencing is by fluorescence microscopy. Flow cytometry can be used for quantitative analysis. In this case, gene silencing is often easier to detect by monitoring the decrease in the mean fluorescence intensity instead of maxgfp expressing cells. 3.2 Initial experimental set-up As the RNAi gene silencing mechanism varies with every cell type, we recommend testing three different amounts of DNA together with a fixed amount of sirna duplex in an initial experiment. For details, see the table below. sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 sample 7 sample 8 sample 9 pmaxgfp 0.5 µg 0.5 µg 0.5 µg 1 µg 1 µg 1 µg 2 µg 2 µg 2 µg sirna µg 1.5 µg* µg 1.5 µg* µg 1.5µg* *Samples 3, 6 and 9 are optional samples meant as control for sirna specificity (negative control). For these samples any unspecific or scrambled sirna can be used.

4 page 4 of Cell culture For detailed information on cell culture conditions and cell numbers please refer to the cell-type specific Optimized Protocol or contact amaxa's Team for further assistance. 3.4 Information about pmaxgfp pmaxgfp encodes the green fluorescent protein (GFP) from copepod Potellina p. The fluorescence intensity of pmaxgfp is comparable to, or slightly exceeds, that of egfp. Esp 3l (7) Eco 31l (18) Nsil (27) Kanamycin Esp 3l (2667) puc ori Kpnl (980) Nhel (988) Eco47lll (993) Agel (997) BspTl (1891) Eco31l (1896) Esp3l (1909) Bglll (1676) Xhol (1680) Sacl (1687) 4 Protocol for suspension cell lines 4.1 Preparation of sirna Preparation of sirna sample Always work in an RNase-free environment. Add 250 µl of the sirna Suspension Buffer to the lyophilized sirna to obtain a 20 µm solution (0.30 µg/µl). Heat the tube to 90 o C for 1 min. Incubate at 37 o C for 60 min. This procedure will disrupt higher aggregates which may have formed during the lyophilization process. It is necessary to maximize sirna silencing potential. You can perform your experiment directly or store sirna stock at -20 o C.

5 page 5 of Nucleofection protocol One nucleofection sample contains 2x10 5-5x10 6 cells 0.5, 1 or 2 µg pmax GFP 1.5 µg sirna (i.e. 5 µl) 100 µl Nucleofector TM Solution Preparation of 1. Cultivate the required number of cells (2x10 5 to 5x10 6 cells). For details see the cell- type specific Optimized Protocol ( 2. Prepare the required amount of sirna and pmaxgfp. 3. Pre-warm the cell-type specific supplemented Nucleofector Solution to room temperature. Pre-warm an aliquot of culture medium containing serum and supplements at 37 C in a 50 ml tube (500 µl per sample). 4. Prepare 12-well plates by filling the appropriate number of wells with 1 ml of culture medium containing serum and supplements and pre-incubate plates in a humidified 37 C/5% CO 2 incubator. 5. Take an aliquot of cell culture and count the cells to determine the cell density. 6. Centrifuge the required number of cells (2x10 5-5x10 6 cells per sample) at xg for 10 min. Discard supernatant completely so that no residual medium covers the cell pellet. 7. Resuspend the pellet in appropriate room temperature Nucleofector Solution to a final concentration of 2x10 5-5x10 6 cells/100 µl. Avoid storing the cell suspension longer than 15 min in Nucleofector TM Solution, as this reduces cell viability and gene transfer efficiency. 8. Mix 100 µl of cell suspension with 1.5 µg sirna (5 µl) and µg pmaxgfp as recommended in table under 3.2 Important: Steps 9-12 should be performed for each sample separately. 9. Transfer the sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap. 10. Select the appropriate Nucleofector TM program (see Nucleofector TM Manual for details). Insert the cuvette into the cuvette holder (Nucleofector TM I : rotate carousel to final position) and press the X button to start the program. 11. To avoid damage to the cells remove the sample from the cuvette immediately after the program has finished (display showing "OK"). Take the cuvette out of the holder. To transfer the cells from the cuvettes, we strongly recommend using the plastic pipettes provided in the kit to prevent damage and loss of cells. Add 500 µl of the pre-warmed culture medium containing serum and supplements to the cuvet- samples Nucleofection

6 page 6 of 8 te and transfer the sample into the prepared 12-well plates. Alternatively, transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37 C heat block. 12.Press the X button to reset the Nucleofector TM. 13.Repeat steps 9-12 for the remaining samples. Cultivation 14.If you have incubated the samples in 1.5 ml microcentrifuge tubes transfer them into post nucleofection the prepared 12-well plates. 15.Incubate cells in a humidified 37 /5% CO 2 incubator. Analyze gene silencing after 24 hours by fluorescence microscopy Protocol for adherent cell lines Preparation of sirna Preparation of sirna sample Always work in an RNase-free environment Add 250 µl of the sirna Suspension Bufferto the lyophilized sirna to obtain a 20 µm solution (0.3 µg/µl). Heat the tube to 90 o C for 1 min. Incubate at 37 o C for 60 min. Perform your experiment or store at 20 o C This procedure will disrupt higher aggregates, which may have formed during the lyophilization process. It is necessary to maximize sirna silencing potential. You can perform your experiment directly or store sirna stock at -20 o C. One nucleofection sample contains 5.2 Nucleofection Protocol 2x10 5-5x10 6 cells. 0.5, 1 or 2 µg pmax GFP. 1.5 µg sirna (i.e. 5 µl). 100 µl Nucleofector TM Solution. Preparation of samples 1. Cultivate the required number of cells (2x10 5 to 5x10 6 cells). For details see the cell type specific Optimized Protocol ( 2. Prepare the required amount of sirna and pmaxgfp. 3. Pre-warm the cell-type specific supplemented Nucleofector Solution to room temperature. Pre-warm an aliquot of culture medium containing serum and supplements at 37 C in a 50 ml tube (500 µl per sample). 4. Prepare 6-well plates by filling the appropriate number of wells with 1.5 ml of culture medium containing serum and supplements and pre-incubate plates in a humidified

7 page 7 of 8 Nucleofection Cultivation post nucleofection 37 C/5% CO 2 incubator. 5. Remove the medium from the cultured cells. Wash cells once with PBS. Aspirate and discard PBS. 6. Harvest the cells, e.g. with trypsin/edta and stop the trypsinization with supplemented culture medium or PBS/0.5% BSA (see Nucleofector TM Manual, for details). 7. Take an aliquot of the trypsinized cell suspension and count the cells to determine the cell density. 8. Centrifuge the required number of cells at 200xg for 10 min. Discard supernatant completely so that no residual medium covers the cell pellet. 9. Resuspend the pellet in appropriate room temperature Nucleofector Solution to a final concentration of 2x10 5-5x10 6 cells/100 µl. Avoid storing the cell suspension longer than 15 min in Nucleofector TM Solution, as this reduces cell viability and gene transfer efficiency. 10.Mix 100 µl of cell suspension with 1.5 µg sirna (5 µl) and µg pmax GFP as recommended in table under 3.2 Important: Steps should be performed for each sample separately. 11. Transfer the sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap. 12.Select the appropriate Nucleofector TM program (see Nucleofector TM Manual for details). Insert the cuvette into the cuvette holder (Nucleofector TM I : rotate carousel to final position) and press the X button to start the program. 13.To avoid damage to the cells remove the sample from the cuvette immediately after the program has finished (display showing "OK"). Take the cuvette out of the holder. To transfer the cells from the cuvettes, we strongly recommend using the plastic pipettes provided in the kit to prevent damage and loss of cells. Add 500 µl of the pre-warmed culture medium containing serum and supplements to the cuvette and transfer the sample into the prepared 6-well plates. Alternatively, transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37 C heat block. 14.Press the X button to reset the Nucleofector TM. 15.Repeat steps for the remaining samples. 16.If you have incubated the samples in 1.5 ml microcentrifuge tubes transfer them into the prepared 6-well plates. 17. Incubate cells in a humidified 37 /5% CO 2 incubator. Analyze gene silencing after 24 hours by fluorescence microscopy.

8 page 8 of 8 This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used as a positive control with this amaxa product only. Any use of the proprietary nucleic acid or protein other than as a positive control with this amaxa product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at license@evrogen.com The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA. # Manufactured by QIAGEN for distribution by amaxa.