SureSilencing Gene Specific siRNA Population
Contents
1. Ehsani A Salvaterra P and Rossi J 2002 Expression of small interfering RNAs targeted against HIV 1 rev transcripts in human cells Nature Biotechnology 20 500 5052. RT Buffer 4 ul 2 5 mM dNTPs 4 ul RNase Inhibitor 1 ul MMLV Reverse Transcriptase 1 ul Final Volume 10 ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step c RT Reaction Add the 10 ul RT Cocktail to the 10 ul Annealing Mixture Mix well but gently with a pipettor and continue incubation at 37 C for 60 min Heat at 95 C for 5 min to hydrolyze the RNA and to inactivate the reverse transcriptase Hold the finished RT Reaction on ice until the next step SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 13 3 PCR Perform one reaction for each RNA sample ddH20 19 5 ul RT Reaction 1 0 ul Buffer PP 1 0 ul 10X Polymerase Buffer 2 5 ul Tag DNA Polymerase 1 0 ul Final volume 25 0 ul Program the thermal cycler for PCR as follows 94 C 3 min 25 cycles of 94 C 15 sec 50 C 15 sec 72 C 30 sec Note DO NOT exceed the recommended number of cycles When the PCR is complete mix 10 ul of each completed PCR reaction with 2 ul of 6X agarose gel loading buffer 4 Product Characterization a Load each sample into separate wells of a 1 agarose gel containing 0 5 ug ml ethidium bromide in 1X TAE b Load an appropriate amount of 100 bp DNA Step Ladder Promega Catalog G695A in an adjacent lane c Electrophorese in 1X TAE at 80V for 40 min d Capture image of gel on a UV Trans Illuminator using a CCD camera or on high speed film e The expected sizes of t
3. Array SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 16 VI REFERENCES 1 Elbashir S Lendeckel W and Tuschl T 2001 RNA interference is mediated by 21 and 22 nucleotide RNAs Genes Dev 15 188 200 2 Nishikura K 2001 A short primer on RNAi RNA directed RNA polymerase acts as a key catalyst Cell 16 415 418 3 Robertson HD Webster RE and Zinder ND 1968 Purification and properties of ribonuclease III from Escherichia coli J Biol Chem 243 82 4 Yang D Buchholz F Huang Z Goga A Chen C Bradsky F and Bishop M 2002 Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells Proc Natl Acad Sci USA 99 15 9942 7 5 Calegari F Haubensak W Yang D Huttner W and Buchholz F 2002 Tissue specific RNA interference in postimplantation mouse embryos with endoribonuclease prepared short interfering RNA PNAS 99 22 14236 6 Chi JT Chang HY Wang NN Chang DS Dunphy N and Brown PO 2003 Genomewide view of gene silencing by small interfering RNAs PNAS 100 6343 6346 7 Semizarov D Frost L Sarthy A Kroeger P Halbert DN and Fesik SW 2003 Specificity of short interfering RNA determined through gene expression signatures PNAS 100 6347 6352 8 Paul CP Good PD Winer and Engelke DR 2002 Effective expression of small interfering RNA in human cells Nature Biotechnology 20 505 508 9 Lee NS Dohjima T Bauer G Li H Li M J
4. SureSilencing siRNA Kit GENE SPECIFIC siRNA POPULATIONS FOR EXPRESSION SUPPRESSION User Manual N SuperArray Bioscience Corporation SuperArray Bioscience Co 7320 Executive Way Suite 101 Frederick MD 21704 Telephone 301 682 9200 1 888 503 3187 Fax 301 682 7300 1 888 465 9859 Web site www superarray com FOR RESEARCH USE ONLY Version 1 1 8 15 2003 SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 A SuperArray Bioscience Corporation SureSilencing siRNA Kit GENE SPECIFIC siRNA POPULATIONS FOR EXPRESSION SUPPRESSION USER MANUAL ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 1 888 503 3187 301 682 9200 e FAX 1 888 465 9859 301 682 7300 e ON LINE ORDER www superarray com e E MAIL order superarray net customer superarray net support superarray net Orders may be placed by fax e mail or from our website Each order should include the following information Caller s contact information 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 http www superarray com SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21704 USA SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 3 TABLE OF CONTENTS l Background and Introduction 4 ll Kit Contents Materials Provided 6 lll Additional Materia
5. acement of the primer relative to the relevant splice junctions The existence of alternative splicing products may not necessarily have been published in the literature By the same token a single band of the expected size from the provided primer set does not necessarily indicate a single splicing product The single band may simply represent the dominant or major splicing product Multiple spicing variants can also produce a single RT PCR band if the amplified fragment lies within a single exon Contamination of your RNA with genomic DNA may also cause the appearance of extra PCR bands What conditions must optimize if using transfection reagents or cell lines different from those recommended by the User Manual We suggest starting to optimize transfection condition using the guidelines provided by the manufacturer of the other transfection reagent itself These mainly include density of the cells at the time of transfection and the mass ratio between the amount of reagent and the amount of nucleic acid used The concentration of the SureSilencing siRNA Populations is 0 4 mg ml You will need to optimize the final transfection conditions yourself Again we find that the Lipofectamine2000 reagent from Invitrogen works the best with SureSilencing siRNA Kits With this product normal growth medium remains on the cells at all times making the usual washing steps and reduced serum conditions that could harm the cells unnecessary Also t
6. for SDS PAGE and Western Verification SuperArray TESTED Biochemical Assays Cells may be left in wells or plates for Cell biological assays such as indirect immunofluorescence Steady state labeling or uptake assays SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 12 Appendix A Use of Separate Components for RT PCR Verification Alternative protocol for those preferring to use components ordered separately 1 Cell Lysis RNA Isolation Total RNA prepared using commercially available kits is suitable for RT PCR analysis to verify silencing of gene expression Total RNA prepared by Trizol Reagent Invitrogen Life Technologies Cat No 15596 018 RNeasy mini kits Qiagen Cat No 74104 or RNAqueous Ambion Cat No 1911 1912 or 1913 gives good results We use the Qiagen RNeasy mini Kits for our QC protocol Total RNA should be dissolved in RNase Free H20 at a concentration of 1 0 mg ml 2 RT Reaction a Prepare the Annealing Mixture For each total RNA sample combine the following into a sterile PCR tube Total RNA 0 5to2 5 ug Random Primers 1 0 ul RNase free H20 to a final volume of 10 0 ul Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C for 3 min Cool to 37 C and keep at that temperature for 10 min b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 37 C 5X
7. he PCR products are listed on the individual product information sheets 5 Image Acquisition and Data Acquisition and Analysis See the same paragraph in the Protocols Section on page 10 SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 14 V TROUBLESHOOTING GUIDE amp FREQUENTLY ASKED QUESTIONS What is the specificity of the SureSilencing siRNA population Buffer SI One potential drawback of SureSilencing approach is the potential for the suppression of closely related genes however several studies suggest that this phenomenon does not greatly complicate siRNA results and analyses 6 7 8 9 Furthermore we are using our previously determined gene specific fragments to generate the siRNA pool Only a subset of the population may represent a closely related gene The population of siRNA duplexes should target the expected gene preferentially because the whole population represents that gene Why are the sizes of my PCR products not the same as the expected size listed on the product sheet Why do I see multiple bands in the RT PCR product We test each primer set using several sources of RNA and using cDNA libraries Under these conditions the RT PCR product is a single band with the predicted and expected size However your RNA experimental sample may contain alternative splicing variants of a particular message Each variant may produce a band of a different size in the RT PCR reaction depending on the pl
8. he SureSilencer siRNA Kit 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 SureSilencer siRNA Kits SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 4 I BACKGROUND AND INTRODUCTION Small interfering RNA siRNA refers to a class of 20 to 25 nucleotide double stranded RNA dsRNA molecules with 2 nucleotide long 3 overhangs Recently Tuschl and colleagues 1 2 showed that siRNA could be used to silence specific genes in mammalian cells Since then these molecules have been used extensively for this purpose However their silencing ability varies While some siRNA can silence gene expression by as much as 80 90 others have no effect Therefore it is essential to test a number of siRNA duplexes to a given mRNA target before finding a functional one Kits have been available for the production of siRNA pools and screening of individual siRNA duplexes however the fragment chosen for use must still be tested and may not work effectively The process can become labor intensive and expensive SureSilencing siRNA pools for gene specific suppression overcome this limitation We take advantage of our already available gene specific cDNA fragments used for our GEArray gene expression profiling systems By incorporating two T7 promoters at both ends of a PCR product 500 800 bp long dsRNA can then be prepared by in vi
9. he nucleic acid may remain with the cells throughout the transfection time course increasing the amount of material able to enter the cells SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 15 Why do not see a difference in the intensities of my RT PCR bands between the control and gene specific siRNA transfected cells If the RT PCR assay used to verify the suppression of expression is not in its dynamic range the difference in MRNA levels will not be apparent The use of too much input RNA or too many PCR cycles can saturate the assay system causing the maximum amount of product to be produced The maximum amount of product produced depends on the assay conditions and not on the amount of specific message in the samples Therefore try to decrease either the amount of RNA or number of cycles or both to find the dynamic range of the assay We do not recommend using more than 25 PCR cycles or more than 2 5 ug of input total RNA The normalization of the intensities of the PCR products to the intensities of the RT PCR product of a housekeeping gene is also very important to the analysis A housekeeping gene is a gene whose expression is not expected to change under the experimental conditions being tested Normalization of expression results to such a gene will control for any differences in the amount of total RNA transferred from your preparation to the assay tube as well as other experimental manipulation variables Ma
10. ls Required 6 IV Protocols A Transfection 7 B RT PCR Verification 8 C Assay Effects of Silencing Gene Expression 11 Appendix A Use of Separate Components for RT PCR Verification 12 V Troubleshooting Guide 14 VI References 16 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 SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray 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 The purchase of SureSilencer siRNA Kit products includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any siRNA pool or primer mix to modify kit components for resale or to use SureSilencer siRNA Kit to manufacture commercial products without written approval of SuperArray Bioscience Corporation No other license expressed implied or by estoppel is granted U S patents may cover certain isolated DNA sequences included in t
11. m temperature Add each 500 ul 100 ul mixture of siRNA and Lipofectamine2000 to its well containing cells and 2 mI 400 ul of normal growth medium Mix gently Incubate the cells at 37 C in a CO2 incubator for 24 48 hours Harvest at least one well per siRNA population for RT PCR verification Use other wells to assay for effects of silencing gene expression Other transfection reagents optimized for siRNA may also be used such as Oligofectamine However SuperArray has tested several reagents and Lipofectamine2000 works optimally with our siRNA populations If you choose to use other transfection reagents follow the protocol as outlined by the reagent manufacturer Assume a stock nucleic acid concentration of 0 4 mg ml in Buffer SI when using other procedures Please optimize these new conditions yourself Note SuperArray Biosciences uses HEK 293 cells for quality control purposes A number of other cell lines including HeLa MCF7 and MDA 231 have been tested with success using a limited number of siRNA products and the above protocol Plating transfection incubation and other conditions may vary with cell type Please optimize these new conditions yourself SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 8 B RT PCR Protocol for Verifying Decrease in Gene Expression Complete kits for RT PCR verification may be purchased from SuperArray Biosciences ReactionReady First Strand cDNA Synthe
12. mponents from other manufacturers listed below a ReactionReady First Strand cDNA Synthesis Kit b ReactionReady PCR master mix 1000 Reagent Product Random Primers Promega Catalog C1181 RNase Inhibitor Promega Catalog N2511 Reverse Transcriptase Promega Catalog M1701 Taq DNA Polymerase Promega Catalog M2661 dNTPs Promega Catalog U1330 C 100 bp DNA Step Ladder Promega Catalog G595A C 01 P 1000A SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 7 IV PROTOCOLS A Transfection The following is a sample protocol for HEK 293 cells using Lipofectamine2000 Invitrogen Catalog 11668 027 019 in a 6 well format Numbers of cells and volumes for using a 24 well format are given in parentheses 1 Note One day before transfection seed 2 5 x 10 5 0 x 104 cells in each well of 6 well plate 24 well plate or in each 35 mm plate with 2 ml 400 ul of growth medium the day of transfection Dilute 5 ul 1 ul of Buffer SI and Buffer NC into separate 250 ul 50 ul volumes of Opti MEM Reduced Serum Medium Gibco and mix gently Make one dilution for each well or plate For each well or plate dilute 5 ul 1 ul of Lipofectamine2000 in 250 ul 50 ul of Opti MEM Mix gently and incubate both dilutions for 5 min at room temperature Combine each diluted siRNA Population with each 250 ul 50 ul Lipofectamine2000 dilution Mix gently and incubate for 20 min at roo
13. ny companies offer a kit containing primers for housekeeping genes We too will soon offer such a kit Perform the RT PCR assay for the housekeeping gene at the same time as the assay for the siRNA targeted gene If using other transfection reagents or cell lines continue to optimize transfection conditions and the RT PCR conditions as described above until evidence of expression suppression in achieved see a decrease in expression at the RNA level as determined by RT PCR however do not see an effect at the level of protein or in my biochemical assay The RT PCR verification will confirm that expression has been decreased at the level of the messenger RNA A change in the RNA level for a particular gene product does not necessarily correlate with a change in the amount of protein in the cell If the protein has a long half life then changes in protein level will take much longer to occur than changes in the RNA level In such a case a modification in the protocol can be attempted After the 24 to 48 hour incubation period re plate the transfected cells at the density recommended in the protocol and repeat the transfection procedure More siRNA reagent will be introduced to the cells and more time will be given for changes in protein levels to occur However this method still does not guarantee that changes in protein level of assay activity will yet be observed The biological system may still limit the ability to see any effects Super
14. rification Verify decrease in message level of specific gene relative to negative control sample AND OR Perform other biochemical molecular or cell biological assay SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 ll KIT CONTENTS MATERIALS PROVIDED BUFFER CONTENTS VOLUME SI SureSilencing siRNA Population YELLOW topped tube 90 ul PP Gene Specific Primer Pair PINK topped tube 50 ul NC Negative Control siRNA Population CLEAR topped tube 15 ul The catalog number of the specific SureSilencing siRNA Kit appears on the tubes of Buffers SI and PP These buffers are specific for each kit Please do not combine with other SureSilencing siRNA Kits The kit contains enough gene specific siRNA population for 18 transfections using 6 well plates or 35mm dishes OR 90 transfections using a 24 well format All components included in this kit should be stored at 20 C and are guaranteed for 6 months from the date received lll ADDITIONAL MATERIALS REQUIRED A For Transfection 1 Transfection reagent We highly recommend Lipofectamine2000 Invitrogen Cat 11668 027 019 Others reagents may also be used however see Notes in the Protocols section 2 Other materials required recommended by transfection reagent manufacturer Opti MEM Gibco B For RT PCR Verification of Gene Expression Knock Down OPTION 1 SuperArray Kits OPTION 2 Individual Co
15. sis Kit C 01 and ReactionReady PCR master mix 1000 P 1000A If using these products follow the protocol described below Individual components for RT PCR may also be purchased separately from other manufacturers See the Additional Materials Required section For your convenience an alternative protocol for RT PCR is provided in Appendix A for using these individual reagents to verify the down regulation of gene expression 1 Cell Lysis RNA Isolation Total RNA prepared using commercially available kits is suitable for RT PCR analysis to verify silencing of gene expression Total RNA prepared by Trizol Reagent Invitrogen Life Technologies Cat No 15596 018 RNeasy mini kits Qiagen Cat No 74104 or RNAqueous Ambion Cat No 1911 1912 or 1913 gives good results We use the Qiagen RNeasy mini Kits for our quality control protocol Total RNA should be dissolved in RNase Free H20 at a concentration of 1 0 mg ml 2 RT Reaction Using SuperArray ReactionReady First Strand cDNA Synthesis Kit a Prepare the Annealing Mixture For each total RNA sample combine the following into a sterile PCR tube Total RNA 0 5to2 5 ug Buffer P Random Primers 1 0 RNase free H20 to a final volume of 10 0 ul Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C for 3 min Cool to 37 C and keep at that temperature for 10 min b Prepare the RT Cocktail This mix
16. the following web page and its links for more information http rsb info nih gov nih image Follow the instructions for that software c Data Analysis During Data Acquisition remember to use the software to determine an appropriate background value That is use the same rectangle or other shape used to surround the gel bands to surround a blank area of the gel and obtain data from that area Subtract that background value from the integrated intensity values of all other data points obtained from the gel bands The intensity of the RT PCR product generated from cells treated with the gene specific siRNA should be less than 30 percent that of the product from control siRNA treated cells SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 11 C Assay Effects of Silencing of Gene Expression There are many ways to characterize the effects on cells brought on by a decrease in the expression of a gene meditated by siRNA The following is a brief list of possibilities Your experiments need not be limited to these suggestions however SuperArray Bioscience has tested some of these applications using a limited number of products Cells may be harvested and RNA isolated for gene expression analysis using ReactionReady Products to verify decrease in gene expression see above GEArray Q Series Gene Array Kits SuperArray TESTED MultiGene 12 RT PCR Profiling Kits Cells may be harvested and protein isolated
17. tro transcription and digested with RNase Ill to produce a population of siRNA duplexes 3 4 5 Since many different siRNAs are included in the population an appropriate siRNA should be present and gene expression can be efficiently supressed As part of the quality control process the SureSilencing siRNA population is quantitated using a spectrophotometer to standardize stock solution concentration and is visualized on a 15 non denaturing acrylamide gel to verify that the toxic long dsRNA was completely digested Every kit contains a population of RNase Ill digested siRNA for a specific gene that has been tested and verified to suppress that gene s expression by at least 70 in mammalian cells A pair of gene specific primers to verify the suppression of gene expression using RT PCR is also enclosed A negative control siRNA pool is also included Features of the SureSilencing siRNA Kit Knock down the expression of a specific gene Pre tested and verified to suppress expression by at least 70 percent Ready to use no enzymatic steps required Includes negative control siRNA Includes primers to verify down regulation by RT PCR SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 Overview of SureSilencing siRNA Kit Procedure Negative Control SureSilencing siRNA siRNA Invitrogen transient transfection 24 to 48 h incubation LipofectAMINE2000 Harvest cells for RNA Perform RT PCR Ve
18. ture can be prepared while the Annealing Mixture is incubating at 37 C Buffer BC 5X RT Buffer 4ul RNase free H20 4 ul RI RNase Inhibitor 1 ul RE Reverse Transcriptase 1ul Final Volume 10 ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 9 c RT Reaction Add the 10 ul RT Cocktail to the 10 ul Annealing Mixture Mix well but gently with a pipettor and continue incubation at 37 C for 60 min Heat at 95 C for 5 min to hydrolyze the RNA and to inactivate the reverse transcriptase Hold the finished RT Reaction on ice until the next step 3 PCR Perform one reaction for each RNA sample Add 150 ul of ddH20 to one tube of ReactionReady PCR master mix 1000 P 1000A Dissolve the lyophilized components by vortexing well For each PCR mix the following components together in a PCR tube ReactionReady PCR master mix 1000 12 5 ul ddH20 10 5 ul RT Reaction 1 0 ul Buffer PP 1 0 ul Final volume 25 0 ul Program the thermal cycler for PCR as follows 94 C 3 min 25 cycles of 94 C 15 sec 50 C 15 sec 72 C 30 sec Note DO NOT exceed the recommended number of cycles 4 Product Characterization a When the PCR is complete load 10 ul of each reaction into separate wells of a 1 agarose gel containing 0 5 ug ml ethidium bromide in 1X TAE NOTE No gel loading dye or buffer is required The ReactionRead
19. y master mix 1000 already contains gel loading dye b Load an appropriate amount of 100 bp DNA Step Ladder Promega Catalog G695A in an adjacent lane c Electrophorese in 1X TAE at 80V for 40 min Note The gel dye runs at a size of 50 bp under these conditions d Capture image of gel on a UV Trans Illuminator using a CCD camera or on high speed film e The expected sizes of the PCR products are listed on the individual product information sheets SuperArray SureSilencing siRNA Kit User Manual Version 1 1 8 15 2003 10 5 Image Acquisition and Data Acquisition and Analysis a Image Acquisition At this time we highly recommend using a CCD camera to capture the image of the gel because the Data Acquisition will be more straightforward and the resulting data will have a wider dynamic range A high speed photograph can be used however the image must be digitized by other means separate software must be used and the dynamic range will be smaller b Data Acquisition If a CCD camera was used to capture the image of the gel the software accompanying the instrument should be able to convert the image of the bands into data Follow the instructions for that software If relying on a high speed photograph of the gel digitize the image of the gel using a desktop scanner to obtain a TIFF file of the image To convert that image into data we then recommend using the software NIH IMAGE Available free from the NIH See
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