Interferon Response Detection Kit User Manual, v.3
Contents
1. 1 degraded or 2 contain an inhibitor Try the reverse transcription again after purifying new RNA If you still do not get sufficient yield try a different RNA purification kit For example Invitrogen s TRIzol Reagent Cat 15596 026 or try using SBI s Full Spectrum Complete Transcriptome RNA Amplification Kit Cat RA101A 1 to increase the amount of starting template Your Reverse Transcription reaction may not have worked well To check this you can run the remaining portion of your reverse transcriptase reaction on a 1 agarose gel containing 0 01 ethidium bromide You should see a smear as shown in Figure 3 If you do not see a relatively bright smear there was a problem with your reverse transcription reaction Do this reaction again and check 3 ul of the product on a gel before continuing with the amplification If you do not see sufficient product again contact SBI Fig 3 Agarose gel showing product of a cDNA synthesis reaction ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 If you see smears or non specific products with your gene specific primers e Confirm that you are using some form of hot start PCR system that ensures specific priming e f you are using hot start and get smearing you may be over cycling your PCR reaction making a mistake in mixing reagents or failing to use optimized PCR reagents e Reduce the number of cycles or use recommende2. Kochs G et al J Biol Chem 2002 277 16 14172 6 Pebernard S lggo R D Differentiation 2004 72 1 9 Persengiev et al RNA 2004 10 12 18 Scacheri et al Proc Natl Acad Sci USA 2004 101 1892 1897 Sledz et al Nature Cell Biol 2003 5 834 839 Stark et al Annu Rev Biochem 1998 67 227 264 Veals SA et al Mol Cell Biol 1992 12 8 3315 24 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual IV Troubleshooting A No Product from cDNA Amplification with Gene Specific Primers If you do not see bands of the expected size for any samples including the Control cDNA and B actin Control Primer Mix One or more of the reagents were omitted during the procedure or the volume of the reactions is incorrect Calibrate your pipette and try amplifying the Control RNA again using primers for B actin There may be a problem with your RT or PCR reagents Replace all the reagents or use commercially available kits for RT and PCR and try to amplify with the control B actin primers If you see bands for the Control cDNA but not for your RNA samples Page 14 You may have less starting RNA than measured Place the amplification reactions back in the thermocycler and perform an additional three cycles 94 C for 30 sec 68 C for 30 sec If after additional cycles there are still no bands or very weak bands compared with the Control RNA reaction your RNA may either be
3. SBI System Biosciences Interferon Response Detection Kit for validation of sIRNA experiments Cat SI300A 1 User Manual Store kit at 20 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 4 082410 contained in this user manual Interferon Response Detection Kit Contents VI Troubleshooting Introduction and Background Overview Detection of Interferon Pathway Activation Variability of the Interferon Response Overview of Protocol sss List of Components mme Additional Required Materials Procedural Guidelines IO MMUNU gt Protocol A Isolating RNA Appendix A Related Products 888 266 5066 Toll Free 650 968 2200 outside US Cat SI300A 1 NNN OP ODM ND 15 15 16 Page 1 System Biosciences SBI User Manual I Introduction and Background A Overview This manual provides details and information necessary to use the Interferon Response Detection Kit to identify and reproducibly confirm non specific stress response of cells to the introduction of SIRNA This kit may also be used for other applications such as for the detection of retroviral infection To ensure optimal results please read the entire manual before using the reagents and material supplied with this system siRNA and Interferon Induction Mammalian hosts have evolved de
4. MX1 ISGF3y IFITM1 MX1 ISGF3y IFITM1 Interferon Induction in HEK 293 Cells 0 75 E control 0 6 m INF induced 0 5 0 4 4 0 3 0 2 01 4 o lt lt r i 8 hours 24 hours INF ELISA pg ml Fig 1 Interferon response detected in two human cell lines MRC 5 and HEK 293 cells were grown to confluency in a T 75 format flask and then transfected with 500 ng ml of a long poly l poly C dsRNA sequence Sigma Cat P9582 Total RNA was prepared from treated and mock transfected control cells at 2 8 24 and 48 hours after transfection The expression levels of OAS1 OAS2 MX1 ISGF3y and IFITM1 genes were determined by end point RT PCR 27 cycles using the protocol described in the kit As is evident from the results shown for MRC 5 cells on the left and HEK293 cells on the right the interferon response was readily detected by examination of the expression levels of these genes Also shown is the level of interferon response in HEK 293 cells as detected by ELISA 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual E Overview of Protocol As can be seen in the flowchart in Figure 2 the procedure for the Interferon Response Detection Kit is straightforward convenient and provides clear measurable results Total RNA 5 Reverse Transcription 1 5 hr Transfer cDNA into tubes with ISG Primers a g Z gt pact rimers IFI
5. a regulatory component Veals S A et al 1992 The ISGF3y Primer Mixture amplifies a 333 base portion of the gene D Variability of the Interferon Response The sensitivity of different cell lines to double stranded RNA activation of interferon induced stress genes and pathways can vary significantly Cell type as well as growth conditions and passage number can affect the susceptibility level and extent of activation of the interferon response The interferon response is readily detected in the numerous human cell lines that activate stress response pathways when double stranded RNA is introduced This response can be clearly seen by the robust increase in OAS1 OAS2 MX1 ISGF3y and IFITM1 expression levels after treatment with concentrations as low as 250 ng ml of a long poly l poly C dsRNA sequence Sigma Cat P9582 in cell lines such as HEK 293 and MRC 5 see Figure 1 Higher concentrations of the double stranded RNA e g 2 5 ug ml can trigger rapid apoptosis and cytotoxicity within 24 to 48 hours Page 4 ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 MRC 5 Cells HEK 293 Cells 2hr 8hr 24hr 48 hr 2hr 8hr 24hr 48hr P actin P actin OAS1 OAS1 OAS2 OAS2 MX1 MX1 ISGF3y ISGF3y IFITM1 IFITM1 HEK 293 Cells E2 hours 7 2 hours m8 hours m8 hours 24 hours 124 hours 148 hours 148 hours Ratio Induced to Control Ratio Induced to Control L o
6. 2 000 bp AmpliSize DNA Ladder Bio Rad Cat 170 8200 e Optional for samples from sources with high RNase activity Ribonuclease Inhibitor SUPERase IN Ambion Cat 2694 H Procedural Guidelines Basal and induced levels of OAS1 OAS2 MX1 ISGF3y and IFITM1 may vary from human cell line to human cell line In addition basal and induced levels of the ISGs may also vary in the same cell line depending on growth conditions Thus it is critical to ensure that you include a non induced control that is treated exactly the same as 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual samples where you introduce siRNA this includes mock transfecting the control cells Since siRNA reagents are delivered to cells via transfection it is crucial to consider the transfection efficiency of the cells when assessing the sensitivity of a particular cell line to the interferon related response to siRNA In addition to the specific procedural notes above be sure to follow these general practices to ensure quality results Page 8 Before dispensing completely thaw all reagents Vortex to mix thoroughly all reagents except for the enzymes After adding reagents to the mixture pipette up and down 5 10 times to ensure mixing Briefly centrifuge each mixture once all the components have been added to ensure there are no reagents left on the sides of the tube separated from the reaction mi
7. TM1 Primers Amplification 1 hr Fl OAST OAS2 MX1 ISGF3y IFITM1 rimers Primers Primers Primers Primers Primers M 4 Fig 2 Flowchart for IRD Kit Procedure Page 6 ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 F List of Components The Interferon Detection Kit provides enough primer in each mix to perform 20 PCR reactions 100 total reactions in 50 ul 20 ul Primer Mixture for OAS1 144 nt 10 uM ea 20 ul Primer Mixture for OAS2 234 nt 10 uM ea 20 ul Primer Mixture for MX1 402 nt 10 uM ea 20 ul Primer Mixture for IFITM1 128 nt 10 uM ea 20 ul Primer Mixture for ISGF3y 333 nt 10 uM ea 20 ul Control Primer Mixture for B actin 110 nt 10 uM ea 6 pl Control cDNA INF induced cells 50 ng ul indicates size of the amplicon The kits are shipped on blue ice and should be stored at 20 C upon receipt Properly stored kits are stable for 1 year from the date received G Additional Required Materials e RNeasy Mini Total RNA Purification Kit QIAGEN Cat 74104 e Reverse Transcription Kit SuperScript III Invitrogen Cat 18080 051 or for small quantities of RNA use SBI s Full Spectrum Complete Transcriptome RNA Amplification Kit Cat RA101A 1 e PCR Reagents Titanium Taq DNA Polymerase Clontech Cat 639208 e Thermocycler with heated lid e 3 Agarose Gel in Tris Acetate EDTA TAE Buffer e DNA Size Ladder with markers from 50 to
8. d reagents As noted the volume of the amplified cDNA added to the gene specific reaction should not exceed 5 V Appendix A Related Products e RNAi Cloning and Expression Lentivectors These FIV and HIV based single and double promoter shRNA and siRNA cloning vectors allow you to clone siRNA templates and efficiently transduce these siRNA constructs in a wide range of cells For a list of currently available vectors please visit our website at http www systembio com B Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual VI Licensing and Warranty Statement Limited Use License Use of SBI s Interferon Response Detection Kit i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Pr
9. dding to a microfuge tube the volume of each of the following components multiplied by the number of first strand synthesis reactions from Part B Amplification Master Mix 30 ul 10X PCR Buffer 6 ul 50X dNTP Mix 6 ul PCR Polymerase 50X 246 ul RNase free Water 288 ul Total volume per first strand synthesis reaction 3 Add 48 ul from the Amplification Master Mix to each of the tubes contain gene specific primers and cDNA from Step 1 4 Place the reactions in a thermal cycler and cycle using the following program e step 1 94 C for 3 min e step 2 68 C for 1 min step 3 94 C for 30 sec 68 C for 30 sec for 20 30 cycles see note e step 4 15 C hold Note You will need to optimize the number of cycles for step 3 of the program depending on the amount of starting RNA Refer to the table below to determine the approximate number of times you should cycle for samples that show a positive stress response to siRNA Starting RNA ng Cycles 300 20 100 23 50 26 25 30 Note Some genes may not normally be expressed or may be expressed at very low levels in some cell types so you may not see a band in some control samples 5 After amplification run 5 ul of each reaction on a 3 agarose gel in 1X TAE Buffer Include a DNA size ladder with markers in the range of 50 2 000 bp e g Bio Rad AmpliSize DNA Ladder You should see results similar to those shown in Figure 1 Depending on your particular RNA sa
10. ersengiev et al 2004 and the cell line The OAS1 NM_016816 and OAS2 NM_016817 1 genes The 2 5 oligoadenylate synthetases OAS represent a family of interferon induced proteins implicated in the mechanism of the antiviral action of interferon When activated by double stranded RNA these proteins polymerize ATP into 2 5 linked oligomers that bind and activate RNase L which plays a significant role in the inhibition of cellular protein synthesis and viral infection resistance Three forms of human OAS have been described Hovnanian A et al 1998 This kit contains primers to detect two of the three forms OAS1 and OAS2 There are two isoforms of the OAS1 gene which share identical N terminal sequence but diverge at exon 7 and have molecular weights of 40 and 46 KDa The Primer Mixture for OAS1 amplifies a 144 base region present in both isoforms Similarly there are two isoforms of OAS2 gene which also share a common 5 region and have molecular weights of 69 and 71KDa The OAS2 Primer Mixture amplifies a 234 base region present in both isoforms The MX1 NM_002462 2 gene Human MX1 protein is a member of the interferon induced myxovirus influenza virus resistance protein family MX proteins and an important component of the innate host defense against RNA viruses The MX family belongs to a superfamily of large GTPases that also includes the dynamins and the interferon regulated guanylate binding proteins MX1 by interactin
11. fense mechanisms against double stranded RNA dsRNA typically present as a consequence of viral infection Upon viral infection cytokine interferon is induced Interferon then activates a signaling cascade culminating in transcriptional activation of hundreds of interferon stimulated genes ISGs Many of these ISGs function in stress response pathways to mediate the cellular antiviral response Haque and Williams 1998 Stark et al 1998 The use of siRNA to selectively knockdown genes of interest has become a powerful tool to study gene function Recently it was discovered that in addition to gene specific silencing siRNA can also induce nonspecific effects in cells by activating ISGs involved in the stress response Bridge et al 2003 Persengiev et al 2004 Scacheri et al 2004 Sledz et al 2003 Pebernard 2004 The observation that treatment of mammalian cells with siRNA molecules can trigger non specific cellular responses raises a serious concern about specificity of RNA interference responses and complicates the interpretation of siRNA knockdown results To address this issue new tools are needed that help confirm the specificity of responses to the knockdown of genes targeted by experimental siRNA and the absence of off target effects induced by the presence of the siRNA molecules SBI s Interferon Response Detection Kit provides an easy and accurate method to ensure that synthetic siRNA or siRNA constructs in plasmid
12. g with a component of the nucleocapsid prevents replication of viral RNA and thereby inhibits the production of new infectious virus particles Kochs G et al 2002 The MX1 Primer Mixture amplifies a 402 nucleotide region of this gene The IFITM1 NM_003641 2 gene Interferon inducible trans membrane proteins IFITMs of approximately 17 kDa have been suggested to play a role in the 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual antiproliferative activity of interferons based on their pattern of induction in interferon sensitive and interferon resistant cell lines and their ability to inhibit cell growth when a membrane fraction enriched in 17 kDa proteins is added to cells in culture IFITM1 has been demonstratively shown to associate with other proteins at the cell surface to form a complex relaying growth inhibitory and aggregation signals Deblandre G A et al 1995 The IFITM1 Primer Mixture amplifies a 128 base fragment of this gene The ISGF3y NM_006084 3 gene Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form This receptor mediated event occurs in the cytoplasm with subsequent translocation of the activated factor to the nucleus ISGF3 has two components termed ISGF3a and ISGF3y ISGF3y serves as the DNA recognition subunit while ISGF3a is the target for interferon signaling and serves as
13. mple or cell type more cycles may be necessary If so perform an additional 3 cycles and check your product again You do not need to add additional PCR Polymerase even if your reaction was cycled overnight as long as you held the reaction at 15 C after cycling You can continue adding two cycle increments up to 30 cycles until you see sufficient product from your amplification reaction 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Note Do not over cycle your PCR reactions to more than 30 cycles because both induced and non induced samples may result in similar band intensity or smear on the gel Alternative Real Time qPCR Procedure Our primers can be used in real time experiments with SYBR Green detection For real time qPCR we suggest TaqMan SYBR Green We recommend using commercially available kits and reagents for SYBR Green qPCR for example Invitrogen s Platinum SYBR Green qPCR SuperMix UDG Cat 11733 038 or QIAGEN s QuantiTect SYBR Green PCR Kit Cat 204143 and following the manufacturer s protocol but increase the extension time to at least 45 seconds Page 12 ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 lll References Bridge et al Nature Genet 2003 34 263 264 Deblandre GA et al J Biol Chem 1995 270 40 23860 6 Haque and Williams Oncol 1998 25 14 22 Hovnanian A et al Genomics 1998 52 3 267 77
14. multiplied by the number of reactions you are processing for each first strand reaction mix Synthesis Master Mix 3 0 ul RNase free Water 2 0 ul 5X Reverse Transcriptase Buffer 1 0 ul 10 mM dNTP Mix 0 5 ul 100 mM Dithiothreitol DTT 0 5 ul Reverse Transcriptase 20X 7 0 ul Total volume per reaction After incubating each of the first strand reactions from step 3 for 10 minutes at 25 C add 7 ul of the Synthesis Master Mix to each reaction tube Incubate the first strand reactions for 1 hour at 42 C then place them at 70 C for 4 minutes The first strand cDNA can be stored at 20 C until you are ready to proceed with gene specific amplification C Gene Specific Amplification Using cDNA This section outlines the procedure to amplify and detect the interferon marker genes using end point PCR and agarose gel electrophoresis Alternatively you can use real time PCR with SYBR Green Details on this alternative procedure are below Note For gene specific PCR we recommend using a hot start PCR polymerase 1 For each first strand synthesis reaction from Part B set up 6 reactions one for each set of gene specific Primers including the B actin control Primers by adding the following 1 0 ul cDNA from first strand synthesis reaction 1 0 ul Gene Specific Primer Mixture Page 10 ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 2 Set up an Amplification Master Mix by a
15. oduct constitutes acceptance of the above terms Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein SBI may have pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited
16. or viral vectors do not induce significant interferon related responses when expressed in the cell system of choice Also the system can be used to assess the effects of reagents such as transfection reagents and other procedural steps that may produce enhance or otherwise affect cellular interferon responses While not all siRNA molecules will activate non specific cellular stress response pathways the only way to confirm the specificity of an siRNA knockdown phenotype or response is to also confirm the absence of Page 2 ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 any nonspecific cellular effects SBI s Interferon Response Detection Kit provides an objective and unambiguous procedure to ensure non specific activation of interferon related pathways do not perturb your siRNA knockdown studies C Detection of Interferon Pathway Activation SBI s Interferon Response Detection Kit uses a PCR based approach to enable researchers to easily measure relative expression levels of five genes involved in the interferon response The use of these five genes helps ensure the kit will accurately detect interferon related stress responses in a variety of cell types and under many different conditions Different cell types may show different levels of increase for these genes Also the level of interferon related stress response that is observed will vary depending on the concentration of the siRNA molecule P
17. tion Although you can often begin to see the increase in some genes in as few as 8 hours see Figure 1 it is advisable to wait 12 to 24 hours to ensure that you get maximal induction of the interferon response marker genes For this procedure start with 1 ug of total RNA at a concentration greater than 0 5 ug ul If the starting amount is significantly less than this we recommend using SBI s Full Spectrum Complete Transcriptome RNA Amplification Kit Cat RA101A 1 to generate enough template for gene specific PCR analysis Part C of this protocol B First Strand cDNA Synthesis 1 For each RNA sample set up a first strand synthesis reaction by adding the following components to a 0 2 or 0 5 ml PCR tube 1 0 ul Random Primer 10 uM not provided 0 5 2 ul 1 ug Total RNA Note Because reagent volumes are small accurate pipetting is critical 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Add sufficient RNase free water to bring the reaction volume up to 3 ul Anneal the primers by placing the tubes in a thermocycler with a heated lid and incubate the reactions at 70 C for 2 minutes Then place the reactions at 25 C for 10 minutes While the reactions are incubating set up a Synthesis Master Mix sufficient for the number of first strand synthesis reactions you are processing This is done by adding to a microfuge tube the volume of each of the following components
18. warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2006 System Biosciences SBI Page 16 ver 3 061023 www systembio com
19. xture When setting up multiple reactions we recommend that you prepare a master mix cDNA is relatively stable and can be stored for a few hours at room temperature or 4 C For longer storage place at 20 C It is important to perform the amplification with the control cDNA provided with this kit The amplified products generated with this control cDNA should be saved and used in subsequent experiments to verify correct amplimer sizes Without this control reaction it will be difficult to troubleshoot any unexpected results ver 3 061023 www systembio com Interferon Response Detection Kit Cat SI300A 1 Protocol A Isolating RNA In general you can use any procedure that provides you with high quality total RNA In our labs the QIAGEN RNeasy Total RNA Purification Kit Cat 74104 provides consistent and reproducible yields If possible confirm the quality of your RNA before starting the amplification The Agilent BioAnalyzer offers a convenient sensitive and reliable method to test small amounts of RNA Alternatively you can run the RNA on an agarose gel to make sure it is intact The average expected yields of total RNA for 1 x 10 cells of some common cell types is listed below Source ug total RNA NIH 3T3 10 HeLa 15 COS 7 35 LMH 12 Huh 15 We recommend that you collect your cells at approximately 24 hours after you transfect or after the addition of components that you want to test for interferon induc
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