SM Now improved with: Si 2 Purfect TRANSFECTION REAGENT PROTOCOL: 2 Si Silencing Duplex Advanced molecule designed for next-generation applications of RNAi. Greater Specificity of Suppression Reliable Platform for Drug Development Effective Knockdown Across Many Cell Types Superior Design and Chemistry Si 2 Silencing Duplex TM www.oligoengine.com
Product: Si 2 Silencing Duplex Scale: 50 nm, 100 nm, 1 µm Format: Lyophylized Duplex Resuspension: Duplex Storage: Resuspend Si 2 Duplex in 250 microliters of endonuclease free water (provided in kit) When resupended in this way, the final salt concentration will be 100mM NaCl, 50mM Tris ph 7.5. For further dilution, use 100mM NaCl, 50mM Tris ph 7.5 Dilution Buffer to avoid duplex denaturation due to low salt conditions. Store dried at -20 C or -80. Resuspended Si 2 Duplex (see protocol) should be stored in small aliquots to avoid multiple freeze thaw cycles. Store at -20 C or -80 C. Your Si 2 Duplex is resistant to nucleases by virtue of the protecting group. A deprotected duplex is significantly more resistant to nucleases than deprotected single strands. However, maintaining sterile, RNAse free conditions is always recommended as a precaution. Transfection Reagent: Purfect Concentration: Purfect Storage: Purfect Stability: 3.0 mg/ml in 80% ethanol Tightly capped at 4 C; DO NOT FREEZE 6 months when stored properly GENERAL INFORMATION Background Cellular uptake of long double stranded RNA (dsrna) has been shown to induce RNA interference in a diverse group of organisms as well as insect cells in culture. RNA interference leads to the inhibition of protein expression by utilizing sequence-specific, dsrna-mediated destruction of the target messenger RNA (mrna). Attempts to induce RNA interference using long dsrna in mammalian cell lines has been met with limited success, due in part to the induction of the interferon response, which results in a general inhibition of protein synthesis. Recently, it has been shown that when short RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mrna can be realized without introducing an interferon response. The Si 2 Silencing Duplex OligoEngine, in recognition of these significant findings, has developed Si 2 Silencing Duplex. The Si 2 Silencing Duplex is actually a novel class of molecules for RNAi that includes, among others, RNA duplexes commonly referred to as short interfering RNA (sirna). Unlike other sirna products, however, the Si 2 Silencing Duplex is not restricted to this specific design or chemistry. Instead, Si 2 products are designed to evolve with advancements in RNAi research. New duplex configurations are continually being tested and developed to increase their efficacy in gene silencing, while eliminating unwanted side effects like off-target suppression. Like sirna, a Si 2 Silencing Duplex acts catalytically at sub-molar ratios to cleave greater than 95% of the target mrna in the cell. The RNA interference effect can be long-lasting and may be detectable after many cell divisions, making the Si 2 Silencing Duplex extremely effective at inhibiting target gene expression once introduced into the cell. 1,2,3 References: 1. Elbashir, S.M. et al. (2001) Nature 411: 494-498. 2. Caplen, N.J. et al (2001) Prot. Natl. Acad. Sci. 98: 9742-9747. 3. Sharp, P.A. (2001) Genes and Development 15: 485-490.
Notes on Transfection Protocol OligoEngine recommends the use of Si 2 Purfect Transfection Reagent for use with the Si 2 Silencing Duplex, and the following protocol. Si 2 Purfect enables highly efficient transfection of Si 2 duplexes, with significantly reduced levels of cell damage as compared to cationic-liposome based transfection reagents. Si 2 Purfect Reagent, when complexed with the Si 2 Silencing Duplex, knocks out target gene expression in a variety of cell lines, including: A549, CHO-K1, COS-7, HEK 293, HeLa,, Hepa1c1c7, HepG2, MCF-7, NIH3T3, RAW 264 and Vero cells. The following protocol is recommended for performing transfections with Si 2 Purfect Reagent in 24-well plates. Each milliliter of Si 2 Purfect Reagent is sufficient in quantity to perform up to 500 transfections in 24- well plates, depending on the specific cell type. When performing transfections in different sized dishes, the amounts of your Si 2 Duplex, Si 2 Purfect Reagent, and culture medium should be scaled up or down in proportion to the surface area of the dish. Transfections are most effective when carried out in complete growth media, with no media change or serum addition required. TRANSFECTION OPTIMIZATION The key to successful transfection is careful optimization of reaction conditions for each individual cell type. The transfection protocol should result in efficient transfection of most cell types; however, to ensure optimal results the following variables should be considered: A. Media Conditions The Si 2 Purfect Reagent yields improved transfection efficiencies when transfections are performed in complete growth medium (instead of serum-free medium) without a post-transfection media change. B. Cell density (confluence) at transfection The recommended cell density for most cell types at transfection is 60-80% confluence. The optimal cell density should be determined for each cell type in order to maximize transfection efficiency. This density should be maintained in future experiments for reproducibility. C. Si 2 Duplex concentration The Si 2 Duplex should be highly purified, sterile, and the correct sequence. The optimal final Si 2 Duplex concentration for transfection should be within the range of 1 to 10 nm in a 24-well plate (1.9 cm2 dish). As a starting point, we recommend using 5nM per 1.9 cm2 dish. In some cases, it may be necessary to use up to 25 nm of your Si 2 Duplex to see maximum inhibition. D. Si 2 Purfect Reagent As a starting point, test three levels of Si 2 Purfect Reagent, such as 0.5 µl, 1.5 µl, 3 µl per well of a 24-well plate, using 25 nm Si 2 Duplex. The optimal Si 2 Purfect Reagent concentration can be determined by titrating the reagent within ranges in Table 1.. The volume of reagent that gives the highest inhibitory effect with the lowest cellular toxicity should be used for future transfections. E. Transfection Incubation Time The optimal incubation time can be determined empirically by testing a range of incubation times from 4-72 hours. The effect of your SI 2 Duplex can be seen as soon as 4 hours post-transfection, but maximum inhibition is often obtained 24-72 hours post-transfection. F. Proper Controls We recommend using a negative control containing serum-free media, Si 2 Purfect Reagent, and a non-specific Si 2 Duplex (found in Oligoengine online catalog). Other controls to consider are cells alone or reagent alone controls. Run such controls in parallel using same volumes and parameters. Table 1. Recommended starting conditions for using Si2 Purfect Transfection Reagent CultureVessel 96-well 48-well 24-well 12-well 6-well 10 cm dish Surface Area* 0.35 cm 2 1.0 cm 2 1.9 cm 2 3.8 cm 2 9.6cm 2 59 cm 2 Serum-free Media 9 µl 25 µl 50 µl 100 µl 250 µl 1500 µl Si 2 Purfect 25 µl/well 25 µl/well 50 µl/well 50 µl/well 100 µl/well 200 µl/well 1µM stock Si 2 Duplex (1-10nM final conc.) 0.125-2.6 µl 1.5-7.5 µl 3-15 µl 6-30 µl 15-75 µl 90-450 µl Complete Growth Media 44 µl 125 µl 250 µl 500 µl 1250 µl 7500 µl
PROCEDURE Dilution of Si 2 Silencing Duplex To dilute your Si 2 Duplex, use 100 mm NaCl, 50 mmtris, ph 7.5, made with RNase-free water. Do not use water alone to dilute the duplex, as this may result in denaturation of the duplex. Transfection of Si 2 Silencing Duplex A. Cell Plating 1. Approximately 24 hours prior to transfection, plate cells at an appropriate cell density (~5x 104 cells/ml in their complete growth medium per 1.9 cm2 well) so that they will be ~40-70% confluent the following day. a 2. Incubate the cells overnight. b B. Complex Formation (perform this procedure immediately prior to transfection) 1. In a sterile, polystyrene 12 x 75 mm tube, add the Si 2 Purfect Transfection Reagent (1 to 5 µl- see Table 1) dropwise into 50 µl of serumfree medium c,d (Opti-MEM I or RPMI 1640 from Gibco BRL are recommended for mammalian cell types). Mix thoroughly by vortexing. 2. Incubate at room temperature for 5-20 minutes. 3. Add Si 2 Duplex (1-25 nm) to the diluted Si 2 Purfect Reagent. Mix by gentle pipetting. 4. Incubate at room temperature for 5-20 minutes. C. Cell Preparation for Transfections in Complete Growth Medium NOTE: For several cell lines tested, we have found that the Si 2 Purfect Reagent yields improved transfection efficiencies when the transfections are performed in complete growth medium (instead of serum-free medium) without a media change following transfection. 1. If necessary, adjust the volume in the well to 250 µl of complete growth media. (see Table 1) 2. Add the Si 2 Purfect Reagent/Si 2 Duplex mixture prepared in step B dropwise to the cells. Gently rock the dish back and forth and from side to side to distribute the complexes evenly. 3. Incubate for 4-72 hours b Note: The above incubation is designed for transfections performed with no media change. If you wish to perform a media change to remove the transfection complexes, incubate the cells for 24 hours, replace the original medium with fresh complete growth medium, and incubate for an additional 24-48 hours. b,e 4. Assay for inhibition of target gene expression. a Since the optimal cell density (confluence) for efficient transfection can vary between cell types, we recommend that you maintain the same seeding protocol between experiments. b Standard incubation conditions for mammalian cells are 37 C in 5% CO2. Other cell types, such as insect cells, require different temperatures and CO2 concentrations. Use conditions appropriate for the cell type of interest. c The Si 2 Purfect Reagent/Si 2 Duplex may form improperly if the transfection medium contains serum, resulting in poor transfection efficiencies. d For transfecting concentrations of Si 2 Duplex beyond 50 nm, or if a precipitate forms upon adding the reagent, use the highest recommended amount of Si 2 Purfect Reagent (see Table 1) and increase the volume of serum-free medium two-fold. e The optimal incubation time should be determined empirically by testing a range of incubation times from 4-72 hr.
TROUBLESHOOTING Low Transfection Efficiency Suboptimal Si 2 Purfect Reagent Determine the optimal Si 2 Purfect Reagent concentration by titrating the reagent from 1 µl to 5 µl per well of a 24-well plate. See Table 1 for recommended starting concentrations. Suboptimal Si 2 Duplex concentration Determine the optimal Si 2 Duplex concentration by titrating from 1 nm up to 25 nm in a 24-well plate. See Table 1 for recommended starting concentrations. Denatured Si 2 Duplex Use recommended buffer (100 mm NaCl, 50 mm Tris, ph 7.5 in RNase-free water) to dilute your Si 2 Duplex. Do not use water as this can denature the Si 2 Duplex. Poor quality of transfecting Si 2 Duplex Avoid degradation or your Si 2 Duplex by using RNase-free handling procedures and plasticware. Degradation can be detected on acrylamide gels. Ensure that the sequence of your Si 2 Duplex is correct for your gene of interest. Fetal calf serum present during Si 2 Purfect Reagent/Si 2 Duplex formation Be sure to use serum-free medium when forming the complexes. Cell density (% confluence) not optimal at time of transfection The recommended cell density for most cell types at the time of transfection is 40-70% confluence. However, you should determine the optimal cell density for each cell type in order to maximize transfection efficiency. Maintain this density in future experiments for reproducibility. Inhibitor present during transfection The presence of polyanions, such as dextran sulfate or heparin, can inhibit transfection. Use transfection medium that does not contain these polyanions. High Cellular Toxicity Si 2 Purfect Reagent/Si 2 Duplex mixture and cells were not mixed thoroughly after adding the complex Mix thoroughly to evenly distribute the complexes to all of the cells. Rocking the dish back and forth and from side to side is recommended. Do not swirl or rotate the dish, as this may result in uneven distribution. Excessive amount of Si 2 Purfect Reagent/Si 2 Duplex mixture was used in transfection Reduce the amount of Si 2 Purfect Reagent/Si 2 Duplex mixture in the transfection. See Table 1 for recommended starting concentrations. Cell density was too low at time of transfection Grow cells to a higher cell density and repeat the transfection. Media change may be necessary If incubating for 48-72 hours, it may be necessary to change the complete media 24 hours post-transfection. SI 2 Silencing Duplex is a trademark of DNAengine, Inc. SI 2 Purfect was developed by Mirus Bio Corporation. Opti-MEM I is a registered trademark of Gibco BRL.
Appendix I Quantifying Your Si 2 Duplex by OD260 Your Si 2 Duplex should be shipped with a datasheet containing the nmoles/µg of each duplex. To perform your own measurements, you can use the following procedure: Supplies Variable wavelength UV spectrophotometer 0.5 ml Quartz cuvettes 1.5 ml microfuge tubes Reagents 1 M Tris-HCl, ph 7.0 RNase free, molecular biology grade water Procedure: 1. Pipet ultra-pure water into the sample so that the final volume is 1.00 ml to provide the Stock Solution. 2. Dilute 10 ml sample into 990 ml of 1 M Tris-HCl ph 7.0 at room temperature to provide the 100X Dilution Sample. 3. Blank the UV spec at 260 nm with 450 ml of 1 M Tris-HCl ph 7.0 at room temperature. 4. Rinse the cuvette with 450 ml of 100X dilution sample, and discard. 5. Fill the cuvette with 450 ml of 100X dilution sample. 6. Record the absorbance reading at 260 nm. 7. Calculations (where 1 ODU = 40 mg of dsrna) Contact Information: SEATTTLE OFFICE Oligoengine, Inc. P.O. Box 95330 Seattle, WA 98112 Phone: (206) 254.0200 Fax: (206) 254.0300 MADISON LABORATORY Oligoengine, Inc. 613 Williamson Street, Suite 208 Madison, WI 53703 Phone: (608) 204.9735 Fax: (608) 204.9736 Toll-Free: 1.800.516.5446 (U.S. & Canada) customerservice@oligoengine.com