User Manual - Biomol GmbH
Contents
1. 1 2 3 4 5 6 7 8 9 10 11 12 A Design1 Txn1 NC Design Txn3 Design1 Txn1 NC Design Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 B Design1 Txn2 Design3 Txn1 Design1 Txn2 Design3 Txn1 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 C Design1 Txn3 Design3 Txn2 Design1 Txn3 Design3 Txn2 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 D Design2 Txn1 Design3 Txn3 Design2 Txn1 Design3 Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 E Design2 Txn2 Design4 Txn1 Design2 Txn2 Design4 Txn1 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 F Design2 Txn3 Design4 Txn2 Design2 Txn3 Design4 Txn2 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 G NC Design Txn1 Design4 Txn3 NC Design Txn1 Design4 Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 H NC Design Txn2 BLANK NC Design Txn2 BLANK P1 P2 P3 P1 P2 P3 Samples in column 1 to 6 are amplified using Samples in column 7 to 12 are amplified PCR primers specific for the GOI using PCR primers specific for the HKG Technical Support 888 503 3187 US 301 682 9200 15 Unless otherwise indicated follow the protocols described in the RT Gene Expression Assays User Manual included with the SABiosciences RT PCR Primer Sets 1 RNA Isolation a Isolate total RNA from each of the 15 transfections b For cultured cells use the RT qPCR Grade RNA Isolation Kit PA 001 Be sure to include the recommended DNase treatment step c Also make sure to perform the RNA quality control described in the RT Gene Expression Assays User Manual 2 Reverse Tr2. NOTICE TO PURCHASER This product is made under license from The Carnegie Institution of Washington However the purchase of this material by a non academic or for profit organization will require a license to use the material from Carnegie License queries may be directed to Gloria Brienza or Gary Kowalczyk at The Carnegie Institution of Washington 1530 P Street NW Washington DC 20005 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SA Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER II The purchase of SureSilencing siRNA includes a limited nonexclusive license to use it for research use only This license does not grant rights to use it for resale or to use SureSilencing siRNA to manufacture commercial products without written approval of SA Bioscience Corporation No other license expressed implied or by estoppels is granted U S patents may cover certain isolated DNA seq
3. 2 12 For 12 RT Profiler PCR Arrays PA 012 24 For 24 RT Profiler PCR Arrays RT Real Time SYBR Green Fluorescein Specifically designed for BioRad iCylcer MyiQ and iQ5 Instrumentation Catalog Number Size PA 011 For 2RT Profiler PCR Arrays PA 011 12 For 12 RT Profiler PCR Arrays PA 011 24 For 24 RT Profiler PCR Arrays RT Real Time SYBR Green Specifically designed for instrumentation that does not require a reference dye BioRad MJ Research Opticon Opticon 2 and Chromo 4 Roche LightCycler 480 System Eppendorf Mastercycler ep realplex Instruments without ROX filter set Catalog Number Size PA 010 For 2RT Profiler PCR Arrays PA 010 12 For 12 RT Profiler PCR Arrays PA 010 24 For 24 RT Profiler PCR Arrays Technical Support 888 503 3187 US 301 682 9200 7 3 RT PCR Primer Assay targeting the suppressed target gene of interest and a housekeeping gene such as ACTB of GAPD to normalize the real time PCR results Find the primer at http www sabiosciences com RT2PCR php PPH00073E 200 RT PCR Primer Set for Human ACTB PPH00150E 200 RT PCR Primer Set for Human GAPDH PPM02945A 200 RT PCR Primer Set for Mouse Actb PPM02946E 200 RT qPCR Primer Set for Mouse Gapdh PPR06570B 200 RT PCR Primer Set for Rat Actb PPR06557A 200 RT qPCR Primer Set for Rat Gapdh 4 RT gPCR Grade RNA Isolation Kit SABiosciences Cat No PA 001 Technical Support support sabiosciences com www sabioscie
4. As free of charge once only You will be asked to provide supporting data demonstrating that the siRNA failed to knock down the target gene by at least 70 at the mRNA level under appropriate transfection conditions Supporting data should include transfection efficiency data quantitative silencing data and data showing 2 70 knockdown of a positive control This offer is valid for up to 6 months after the date of delivery Technical Support support sabiosciences com www sabiosciences com 4 How It Works Mix siRNA with SureFECT transfection reagent ALLE Do reverse transfection for your cells 24 48 hours shRNA3 shRNA 4 Negative a Ad Isolate total RNA for checking knock down efficiency or for your phenotype assay Verify Suppression of Gene Expression Using Real Time PCR Perform biochemical cell or molecular biological assay of gene function Figure 1 Overview of SureSilencing Synthetic siRNA Procedure Technical Support 888 503 3187 US 301 682 9200 5 Il Kit Contents and Related Information A siRNA Set Contents Component l Specification Quantity siRNA ee siRNA 12 5nmol Four 4 a a one 1 B siRNA individual Content Component Specification Quantity SIRNA R EG siRNA 12 5 nmol One 1 on a one 1 Resuspension Instruction Resuspend lyophilized siRNA duplex in 625 ul RNase free ddH2O to get a 20 uM stock solution Aliquots
5. S SABiosciences walgmanrt 2 C biomol info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D User Manual SureSilencing siRNA Genome Wide High Purity Synthetic RNA Interference See Purchaser Notification for limited use license and warranty information pages 2 and 4 Part 1043A Version 1 1 03 24 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA 1 TM SureSilencing siRNA Genome Wide High Purity Synthetic RNA Interference BIOMOL GmbH Waidmannstr 35 22769 Hamburg O biomol info biomol de www biomol de S e r d n u d Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D For Catalog Numbers SIH X SIMHHHHHHX SIRHHHHHHX Ordering and Technical Service Contact Information e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www sabiosciences com e E MAIL order sabiosciences com to place an order support sabiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www sabiosciences com
6. anscription First Strand cDNA Template Synthesis a Perform one reverse transfection for each of the 15 total RNA samples one per transfection a Follow the instructions in the RT Gene Expression Assays User Manual included with the RT PCR Primer Sets b For convenient pipetting below dilute each completed 20 ul RT reaction 50 fold by adding 980 ul of ddH20 3 Primer Set and Master Mix Cocktail For real time PCR prepare two separate cocktails one for the GOI and one for the HKG using the following recipe Component Volume 2X SABiosciences Real Time PCR Master Mix 625 ul ddH20 75 yul RT primer set for GOI or HKG 50 ul Final Volume 750 ul 4 Set Up the Reactions Add 15 ul of the appropriate primer set and master mix cocktail and 10 ul of the appropriate diluted cDNA template RT reaction to the appropriate PCR wells as outlined in Table 1 5 Perform Real Time PCR Perform PCR as described in RT Gene Expression Assays User Manual included with the RT PCR Primer Sets Technical Support support sabiosciences com www sabiosciences com 16 6 Data Analysis An Excel based data analysis template that automatically performs the calculations below is available for download from our website at the following address http www sabiosciences com rnaidataanalysis php a Separately determine the average of the technical triplicate PCR C values and their standard deviations for both genes in
7. are recommended when repeated use is needed Storage Conditions All components included with this catalog number are shipped in ambient temperature Store lyophilized siRNA duplex at 20 C or below Stable for at least 6 month from the date of shipment Once resuspended store at 20 C or below in non frost free freezer avoid contact with RNases and repeated freeze thaw cycles Technical Support support sabiosciences com www sabiosciences com 6 lll Additional Materials Required A For Transfection We recommend using SureFECT Transfection Reagent SABiosciences Cat No SA 01 B For Support Reagents e siRNA Transfection Reagent SureFECT Transfection Reagent SA 01 e Negative Control siRNA SIH165860A SIM166769A SIR766773A e RT PCR Gene Specific Primer Please check our website at http Awww sabiosciences com RT2PCR php e RT PCR House Keeping Gene Primer Please check our website at http www sabiosciences com QRTsearch php target housekeeping B For Real Time RT PCR Verification of Gene Suppression 1 RT First Strand Kit Catalog Number C 03 2 SABiosciences RT Real Time SYBR Green PCR Master Mix Be sure to pick the correct one for the instrumentation in your laboratory RT Real Time SYBR Green ROX Specifically designed for All ABI and Stratagene Instrumentation Eppendorf Mastercycler ep realplex Instruments with ROX filter set Catalog Number Size PA 012 For 2RT Profiler PCR Arrays PA 01
8. cription reaction and only dilute the Technical Support 888 503 3187 US 301 682 9200 13 completed reaction by four fold adding 60 instead of 100 ul of ddH20 but still use 10 ul of the dilution to setup the PCR 4 Real time PCR analysis not performed properly Make sure that real time PCR analysis was set up and performed properly Consult the Troubleshooting Guide of the RT Gene Expression Assay User Manual if using the RT PCR Primer Sets and RT Real Time SYBR Green PCR Master Mixes from SABiosciences If using other reagents for real time PCR consult the original manufacturers recommendations and suggestions Note Only use real time PCR to determine the level of knock down No other RNA detection method e g Northern Blot or conventional PCR will be quantitative enough to observe a 70 percent knockdown Western Blot is also unreliable because the success of knockdown at the protein level also depends on the quality of the antibody and the biological half life of the protein whereas RNA interference specifically acts at the RNA level See below C The expression at RNA level is decreased but no decrease is observed at the level of protein or biochemical assay The RT PCR verification will confirm that expression has been decreased at the level of messenger RNA and that the SureSilencing siRNA functioned correctly A change in the RNA level for a particular gene does not necessarily immediately correlate with a change in
9. each of the replicate transfections of each design and the negative control b Separately calculate individual AC values for each biological replicate transfection of each design and the negative control GOl Specific shRNA AC AVG GOlI Specific shRNA C GOI AVG GOl Specific shRNA C HKG GOl Specific siRNA AC STDEV STDEV 4 STDEV sige Negative Control shRNA AG AVG Negative Control shRNA C GOI AVG Negative Control shRNA C HKG Negative Control shRNA AC STDEV STDEVZ STDEV pc c Calculate the average AC and its standard deviation across the biological replicates for each design d Calculate the average AAC and its standard deviation for each design AA C Gene Specific snRNA AC Negative Control snRNA AC AA C STDEV STDEV2 STDEV ene Specific ACt NegativeControl ACt e Calculate the average knockdown and its 95 confidence interval Percent Knockdown 100 100 x 2 AAC Lower 95 Confidence Interval Boundary 100 100 x 2 AAC AA C STDEV Upper 95 Confidence Interval Boundary 100 100 x 2 AAC AA Ci STDEV f Interpretation Successful Design Observed KD gt 70 and an upper 95 C I boundary gt 55 5 Failed Design Observed KD lt 33 3 and a lower 95 C I boundary lt 55 5 Two out of the four designs should be successful Use at least two of the four pre designed siRNAs that demonstrate the greatest percent knock down
10. ells macrophages or other cell lines and even animals with different methods For these applications we recommend finding relevant references describing proven methods before beginning your experiments Technical Support support sabiosciences com www sabiosciences com 12 VI Troubleshooting Guide A Low transfection efficiency 1 Transfection efficiency primarily depends on the cell line used Therefore it is very important to optimize the transfection conditions for each cell type under study Variable details to consider when optimizing the transfection conditions include cell density cell viability amount of siRNA ratio of siRNA to transfection reagent transfection complex formation time and transfection incubation time see the detailed protocols for our recommendations 2 Use our positive control siRNA to optimize transfection efficiency 3 If using the FAM labeled SureSilencing siRNA Observe the cells carefully with fluorescence microscope under high power field Check the transfected FAM positive cells and the total number of cells nuclear DNA stain in the same fluorescent view of the same microscope field Be sure to obtain numbers from several different randomly chosen microscope fields in the interior not toward the edges of the cell culture well The labeled siRNA signal is not as strong as EGFP signal you may ever see The wash is necessary before observation in order to lower the background Observations for l
11. in your subsequent gene function assays and studies Technical Support 888 503 3187 US 301 682 9200 17 SureSilencing siRNA User Manual Part 1043A Version 1 1 3 24 2009 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com
12. ive control siRNA transfected cells using an optimized siRNA delivery condition Please follow the recommendations in this User Manual to ensure the optimal level of knockdown If more than two of the siRNAs in one set fail to knock down target gene expression please contact our Technical Support to discuss your results and be prepared to provide your results in an Excel file as an email attachment If a product failure is verified we will send you another set of four pre designed siRNAs for free What transfection method should I use with the SureSilencing siRNA We recommend using the SureFECT transfection reagent SABiosciences Cat No SA 01 For the majority of cell lines tested it is an exceptional reagent providing superior combination of maximal transfection efficiency and minimal cytotoxicity If you have a previously optimized siRNA transfection method for your cell line you may use that method to transfect the SureSilencing siRNA To quickly optimize transfection conditions use a system that encodes an easily screening marker such as GAPDH based enzyme activity assay If your cells do not transfect well with lipid based or chemical based methods electroporation method such as those provided by Lonza Amaxa may be a useful alternative Can use the SureSilencing siRNA with primary cells or macrophages or for injection into live animals Yes there are many publications that have shown the efficient delivery of siRNA into primary c
13. ll represent guidelines and may need to be optimized Note 30 nM 30 nmol L 30 pmol mL 30 fmol uL Traditional Forward Transfection Protocol This is just a general guideline for 96 well transfection the optimal conditions amounts should be determined for each new cell line cell type being transfected a One day before transfection seed 6000 cells in each well of 96 well plate with 200 ul of growth medium b On the day of transfection mix 2 pmol 2 ul of each gene specific siRNA or the negative control siRNA into 38 ul of the above Opti MEM Medium diluted SureFECT total volume 40 ul Mix gently Prepare separate mixtures for each replicate c Incubate for 20 min at room temperature and add 40 ul of the siRNA SureFECT mix to each well Add 160 ul of regular fresh media to each well Mix gently e Incubate the cells at 37 C in a CO2 incubator for 24 to 72 hours Determine the target gene knock down at 48 hours recommended after transfection with real time PCR g Determine the phenotype change at the time point you optimized for your experiments ox h Our rigorous real time RT PCR protocol for verifying suppression by RNA interference relies on triplicate transfections for each gene specific siRNA design and the negative control siRNA for statistically significant results See the Appendix of the User Manual for more details Other siRNA delivery method protocols For using other siRNA delivery me
14. mined for each new cell line cell type being transfected a On the day of transfection dilute 0 3 ul SureFECT with 37 7 ul Opti MEM medium for each well the optimized condition for different cell line should be evaluated by customer b Mix 2 pmol 2 ul of each gene specific siRNA and the negative control siRNA into 38 ul of the above Opti MEM diluted SureFECT total volume 40 ul Mix gently Prepare separate mixtures for each replicate c Incubate 40 ul of the siIRNA SureFECT mixture in each well for 20 min at room temperature d Add 160 ul cell suspension prepared during the incubation step at 8000 12000 cells 160 ul in regular culture media to each well of 96 well plate Mix gently e Incubate the cells at 37 C in a CO2 incubator for 24 to 72 hours f Determine the target gene knock down at 48 hours recommended after transfection with real time PCR g Determine the phenotype change at the time point you optimized for your experiments Technical Support 888 503 3187 US 301 682 9200 9 Plating Surface 2 uM Serum Total Plating cell Format Area siRNA SURET free volume number Medi wells plate cm well ul ul ma ul per well 96 0 3 3 0 3 40 200 10000 24 2 9 1 5 100 600 50000 12 4 18 3 150 1200 1000000 6 10 36 6 200 2400 200000 Table 1 Recommended volumes for transfecting 30 nM siRNA in various plating formats SureFECT volumes and other conditions per we
15. nces com 8 IV Protocol A Transfection We recommend the use of SureFECT transfection reagent SABiosciences Cat No SA 01 For virtually all cell lines tested SureFECT is an exceptional transfection reagent providing maximal transfection efficiency and minimal toxicity If you have already optimized a transfection reagent and protocol for your cell line of interest you may use that protocol to transfect the SureSilencing siRNA into the same cell line Just be sure that the original protocol optimization used siRNA and assayed percentage of mRNA or protein knockdown or functional inhibition We recommend using reverse transfection protocols with the SureFECT transfection reagent This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods SureFECT will also work well as a reagent for traditional forward transfection methods The following protocols are written on a per well basis and are designed for the transfection of an adherent cell line Hela ATCC with the SureFECT transfection reagent using a 96 well cell culture plate We recommend that you set up three 3 replicate transfections for each of the four gene specific siRNAs and the negative control SureSilencing siRNA using an optimized transfection protocol Reverse Transfection Protocol This is just a general guideline for 96 well transfection the optimal conditions or amounts should be deter
16. ong time may bleach the FAM signal NOTE DO NOT try to use the phase cell and fluorescence GFP views separately to estimate or count the total number of cells and the number of transfected cells respectively B Knockdown not distinguishable by real time PCR 1 Poor transfection efficiency Make sure that your transfection efficiency is optimized You must be looking at a population of cells that is nearly 100 percent transfected for an accurate determination of knockdown 2 Poor real time PCR reproducibility Make sure that your triplicate real time PCR threshold cycle values demonstrate a high degree of reproducibility with a standard deviation of roughly 0 25 to 0 33 cycles A seventy 70 percent knockdown of expression will be observed as only a 1 74 difference in normalized C values for negative control vs gene specific siRNA transfected cells This specific level of reproducibility is required to reliably detect such a difference 3 Low level of expression of gene of interest To accurately determine knockdown by real time PCR the level of expression of the gene of interest in control or un transfected cells should be at least reasonably well expressed with a C value less than 30 Real time PCR cannot determine the relative expression of genes expressed at a lower level C gt 30 with enough reproducibility to detect a seventy 70 percent knockdown of expression Try using more input RNA up to 5 ug in the reverse trans
17. the amount of protein in the cell If the protein has a long half life protein level change will take much longer to occur than RNA level change Technical Support support sabiosciences com www sabiosciences com 14 Appendix Real time RT PCR Protocol for Verifying Suppression A detailed description of the theory behind RNA interference validation using real time PCR may be found in our white paper entitled Did Your RNAi Experiment Work Reliably Validating RNA Interference with Real Time PCR For statistically significant results the method relies on triplicate transfections for each gene specific siRNA design and the negative control siRNA It also requires triplicate real time PCR reactions to characterize the targeted gene of interest GOI and a housekeeping gene HKG to normalize the results using the total RNA sample from each transfection Typical housekeeping genes include B actin and GAPDH Table 1 Setting up real time PCR validation of suppression The triplicate reactions for each gene in all five triplicate transfections may be conveniently setup in a 96 well PCR plate as depicted in this table The table represents a 96 well plate The reactions in the first set of six numbered columns will characterize expression of the GOI in the indicated RNA samples while the second set of six numbered columns will characterize expression of the HKG in the corresponding RNA samples
18. thods such as electroporation please refer the manufacturer s protocol for your cell line Technical Support support sabiosciences com www sabiosciences com 10 C Assay Effects of Silencing Gene Expression There are many ways to characterize the cellular effects caused by a decrease in the expression of a specific gene meditated by RNA Interference The following is a brief list of possible ways However your experiments do not need to be limited by these suggestions Cells may be harvested and RNA isolated for gene expression analysis using SABiosciences RT Profiler PCR Arrays SABiosciences RT Real Time PCR Primer Sets and SYBR Green Master Mixes SABiosciences GEArray Focused DNA Microarrays Cells may be harvested and protein isolated for SDS PAGE and Western Verification SABiosciences Multi Analyte Profiler ELISArray Kits SABiosciences Single Analyte ELISA Kits Biochemical Assays Cells may be left in wells or plates for SABiosciences Cellular Activation Signaling ELISA CASE Kits Cell biological assays such as morphology and immunofluorescence staining Technical Support 888 503 3187 US 301 682 9200 11 V SureSilencing siRNA FAQs What is the SureSilencing siRNA guarantee We guarantee that at least two of the set of four SureSilencing siRNA will knock down the expression of target gene by at least 70 percent in transfected cells as measured by real time qRT PCR relative to negat
19. uences included in the SureSilencing siRNA Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using SureSilencing siRNA CONTENTS l Background and Introduction 4 Il Kit Contents 6 III Additional Materials Required T IV Protocol 8 A Transfection 8 B Assay Effects of Silencing Gene Expression 10 V SureSilencing siRNA FAQ 11 VI Troubleshooting Guide 12 Appendix Real time RT PCR Protocol for Verifying Suppression 14 Technical Support support sabiosciences com www sabiosciences com 3 I Background and Introduction RNA Interference a now commonplace and popular method for exploring gene function suppresses the expression of a specific gene of interest in transformed mammalian cell culture or even in animal model Upon suppression missing or altered activities in the cell can be attributed to the function of the affected gene The most commonly used technique small interfering RNA siRNA is useful for many applications and has been proved to work optimally in many cells with high efficacies SureSilencing siRNA are designed using an experimentally validated algorithm These synthetic double strands RNA specifically knock down the expression of specific genes by RNA interference Each synthetic siRNA duplex is HPLC purified to provide the maximum amount of full length strand to target your gene of interest in full power Our e
20. xperimentally verified siRNA design algorithm assures gene knockdown specificity and efficacy An advanced specificity search in addition to BLAST built into the algorithm helps to reduce the potential off target effects At least two of the 4 provided SureSilencing siRNAs in each set is guaranteed to knock down the expression of target gene at the mRNA level by at least 70 percent in well transfected cells Benefits of the SureSilencing synthetic siRNA High Purity with low price All of our synthetic siRNA is HPLC purified that provides high quality siRNA to increase knockdown efficacy and lower off target effects GUARANTEED Knock down the expression of any targeted human mouse or rat gene by at least 70 percent with the full set order Control for non specific and off target effects Convenient with customization opportunity People can order the full set or individual one for their preference If customer has their own design in hand customized synthesis is also available At least two of the four provided pre designed SureSilencing siRNA are guaranteed to knock down the expression of target gene at mRNA level by at least 70 percent as measured by real time qRT PCR in transfected cells as described in this User Manual SureSilencing siRNA comes with a one time only replacement offer If a set of SureSilencing siRNAs is ordered and less than 2 of the siRNAs result in gene silencing SABiosciences will provide 4 additional siRN
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