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1. NOTE These constructs are transfection grade and are ready for transient transfection These constructs are specifically designed to inhibit transformation and are NOT MEANT for introduction and amplification in bacteria Each Cignal Finder 10 Pathway Reporter Array includes the following components e Tubes 1 to 10 contain 10 different Cignal Pathway Reporters 100ng ul 2 X 50 ul e Tube 11 contains Cignal Reporter Assay negative control 100ng ul 2 X 50 ul e Tube 12 contains Cignal Reporter Assay positive control 100ng ul 2 X 50 ul Each strip is numbered from 1 to 12 Please refer to the specific Cignal Finder Array product specification sheet for the identity of each individual reporter assay in that array The strip cap used for each 12 tube strip has a tab attached to the cap for tube 1 This assists you in maintaining the caps in the proper orientation Technical Support support SABiosciences com www SABiosciences com 6 Version 1 5 B Description of Individual Cignal Reporter Assays Each Cignal Reporter Assay Kit includes the following components 1 Reporter Each reporter is a mixture of an inducible transcription factor responsive construct and constitutively expressing Renilla luciferase construct 40 1 The inducible transcription factor responsive construct encodes the firefly luciferase reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transc
2. Reporter Assay 13 D Co transfection Protocol for shRNA Reporter Assay 15 E Co transfection Protocol for Expression Vector Reporter Assay u F Transfection and Treatment Protocol for Reporter Assay 20 Small Molecules Organic Compounds G Transfection and Treatment Protocol for Reporter Assay 23 Peptide Recombinant Protein H Scaling up Transfection Experiments 26 Appendix Cignal Finder 10 Pathway Array Product Descriptions 27 Technical Support support SABiosciences com www SABiosciences com 3 Cignal Finder Reporter Arrays tube format Introduction The Cignal Finder 10 Pathway Reporter Arrays enable you to pinpoint the pathways regulated by the gene products or chemical compounds being studied in your laboratory The Cignal Finder Arrays consist of 10 dual luciferase reporter assays and are designed for use in one of four research areas The targeted research areas are cancer immunology development and toxicology In this era of post genomics life science research many labs are investigating how diverse signal transduction pathways function on their own and in combination within the cell The Cignal Finder Arrays equip life science researchers to carry out such studies with speed and confidence The arrays are delivered in 12 tube strips including important negative and positive controls The assays are used right out of the box for the transfection or reverse transfection of the reporter assay
3. Cat No SA 01 as a transfection reagent The Cignal Reporter Assay however also performs equally well with other transfection reagents such as Lipofectamine 2000 Invitrogen Cat No 11668 027 or FUGENE 6 Roche Cat No 1815091 When using alternative transfection reagents please refer to the manufacturer s instructions on the use of those reagents Optimization of transfection conditions The sensitivity of the Cignal Reporter Assay depends on the transfection efficiency The transfection efficiency in turn primarily depends upon cell line used Therefore it is very important to optimize the transfection conditions for each cell type under study Variables to consider when optimizing the transfection conditions include cell density cell viability amount of DNA ratio of DNA to transfection reagent transfection complex formation time and transfection incubation time see the detailed protocols for our recommendations The positive control construct included with each Cignal Reporter Assay can be used for determining the optimal transfection conditions Optimization of assay condition The response rate in the Cignal Reporter Assay depends on the assay conditions conditions of the experimental treatment To obtain maximum response given by any stimulus perform dosing and time course studies The optimal amount of stimulus and the time of treatment must be obtained empirically for each experiment see different protocols for our
4. with 1X 10X and 100X amount of small molecule or organic compound 1X is the lowest appropriate amount of small molecule or organic compound expected to modulate the signaling pathway 11 To study the effect of small molecule or organic compound we recommend harvesting cells 6 hours or 18 hours after treatment to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We found some components in DMEM interfere with the SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 22 Version 1 5 G Transfection and Treatment Protocol for Reporter Assay Peptide Recombinant Protein The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The C
5. 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 3 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs snARNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct snRNA SureFECT complexes This give
6. 5 of fetal bovine serum to each well containing constructs siRNA SureFECT complexes This gives a final volume of 150 pl Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 14 Version 1 5 D Co transfection Protocol for shRNA Reporte
7. 0 3 ul 25 ul 6 ta a 200 ng 25 ul 0 3 ul 25 ul 7 fa a 100 ng ee 25 ul 0 3 ul 25 ul 8 ao A 200 ng 25 ul 0 3 ul 25 ul 9 ve a 25 ul 0 3 pl 25 ul Carrier DNA means any empty plasmid such as a pUC or a pBR plasmid 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 888 503 3187 US 17 Technical Support 301 682 9200 Cignal Finder Reporter Arrays tube format Add 25 ul of Opti MEM to each of 9 polystyrene tubes avoid using DMEM along with the following Experimental transfections 1 ul 100 ng Cignal reporter 100 ng experimental vector expressing gene of interest 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng experimental vector expressing gene of interest Control transfections 1 ul 100 ng Cignal reporter 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng negative control expression vector 1 ul 100 ng Cignal negative control 100 ng experimental vector expressing gene of interest 100 ng carrier DNA 1 u
8. 8 500 3 2 1000 250 000 2 X 100 20 pmol 1 5 ug 6 well 9 4 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 pg 35 mm 8 0 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 pg 6 60mm 21 2000 18 0 5000 1 5X 10 2 X 500 100 pmol 8 0 pg 6 100mm 55 5000 45 0 15000 15ml 4 5X 10 2 X 1500 300 pmol 25 pg a 2x means one volume of Opti MEM medium for diluting constructs and another volume of Opti MEM medium for diluting SureFECT For any other troubleshooting or technical questions about the Cignal Reporter Assay please call one of our Technical Support representatives at 1 888 503 3187 or 301 682 9200 or email at support SABiosciences com Technical Support support SABiosciences com 26 www SABiosciences com Version 1 5 LIMITED PRODUCT WARRANTY 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 SABiosciences 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 Luciferase Limited Use Label License READ THIS FIRST BEFORE OPENING PRODUCT Firefly and or Renilla Luciferase and Monster Green Limited Use Label License For research use only The terms of the l
9. A SABiosciences A QIAGEN Company User Manual ee Cignal Finder 10 Pathway Reporter Arrays tube format Cell Based Multi Pathway Activity Assays See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1034A Biosciences co Srsion Ll Executive wat A201 vo 21703 USA Cignal Finder 10 Pathway Reporter Arrays tube format Cell Based Multi Pathway Activity Assays User Manual For Catalog Numbers CCA 00 L 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 SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Introduction 4 Il Product Contents and Descriptions 6 Ill Additional Materials Required 9 IV Protocol 10 A Before you begin 10 B Generalized Transfection Protocols 11 C Co transfection Protocol for siRNA
10. MEM Reduced Serum Medium Invitrogen Cat No 31985 062 Fetal bovine serum FBS Non essential amino acids NEAA Invitrogen Cat No 11140 050 Penicillin Streptomycin Hemacytometer Dual Luciferase Assay System o Dual Luciferase Reporter Assay System Promega Cat No E1910 This system requires cell lysis and is well suited for the rapid quantitation of both luciferase reporters when using luminometers with reagent auto injectors o Dual Glo Luciferase Assay System Promega Cat No E2920 This system is used to assay for both luciferase reporters on intact cells in growth medium This system can be used with any luminometer including those without reagent auto injectors 96 well white opaque flat bottom microtiter plate Luminometer Technical Support 888 503 3187 US 301 682 9200 9 Cignal Finder Reporter Arrays tube format IV Protocol A Before you begin 1 Cell line selection The Cignal Reporter Assay may be used with various mammalian cell lines Cell lines show a great deal of variation in the levels of signaling proteins The transcriptional activator activities in the cell line used will determine the sensitivity of the assay A cell line should be selected based on the functionality of the signal transduction pathway under investigation as well as for the transfectability of the cell line see below Transfection reagent selection We recommend the use of SureFECT SABiosciences
11. T transfection per well Control Control per well siRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 ul 2 pmol 25 ul 0 3 pl 25 ul 100 ng 2 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 pl 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 2 pmol sequence specific siRNA Control transfections 1 ul 100 ng Cignal reporter 2 pmol negative control siRNA 1 ul 100 ng Cignal negative control 2 pmol sequence specific siRNA 1 ul 100 ng Cignal negative control 2 pmol negative control siRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently Technical Support 888 503 3187 US 301 682 9200 13 Cignal Finder Reporter Arrays tube f
12. ansfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 6 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 COz Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specif
13. e are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 25 Cignal Finder Reporter Arrays tube format H Scaling up transfection experiments To transfect cells in different tissue culture formats vary the amounts of constructs number of cells and volume of SureFECT and medium used in proportion to the surface area as shown in the Table 7 The parameters shown in Table 7 are standardized for HEK 293H cells Use these parameters as a starting point to optimize transfections for your cell line of interest Table 7 Reagent amounts for transfecting cells in different size culture vessels Type of Surface Starting Starting Volume of Starting Volume of siRNA shRNA Plate Area amount of Volume of Cell No of Opti MEM Vector or Gene cm per construct SureFECT Suspension Adherent Medium Expression Vector well ng well ul well ul well Cells per ul per Well Well 96 well 0 3 100 0 3 100 20 000 2 X 25 2 pmol 200 ng 48 well 0 95 150 0 8 250 62 500 2X50 5 pmol 500 ng 24 well 1 9 250 1 6 500 125 000 2X50 10 pmol 750 ng 12 well 3
14. error when setting up transfection cocktails steps 1 through 4 Technical Support support SABiosciences com www SABiosciences com 20 Version 1 5 Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 ul Opti MEM 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 5 4 Mix gently and incubate for 20 minutes at
15. es com 12 Version 1 5 C Co transfection Protocol for siRNA Reporter Assay The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors If you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for optimizing transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be optimized according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 2 Guidelines for setting up co transfections of siRNA and Cignal Reporter Assays Table 2 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive siRNA Control Nucleic per well SureFEC
16. he effect of small molecules organic compounds Table 5 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Small Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Molecule DNA diluent per well SureFECT Transfection per well Control Control Organic per well diluent hours per well Construct Compound per well per well per well 100 ng 1 1 0 pl 25 ul 0 3 ul 25 ul 100 ng a 2 1 0 ul 1X 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 10X 25 ul 0 3 pl 25 ul 4 100ng 100X 25 ul 0 3 ul 25 ul 1 0 pl H Hp H 100 ng 5 1 0 ul 25 ul 0 3 pl 25 ul 30 hor 42h 100 ng 6 1 0 ul 1X 25 ul 0 3 pl 25 ul 100 ng 7 1 0 nl 10X 25 ul 0 3 ul 25 ul 100 ng 8 1 0 ul 100X 25 ul 0 3 pl 25 ul 100 ng 9 1 0 ul 25 ul 0 3 pl 25 ul 41X is a smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor
17. hor 42h 6 ee i 1X 25 ul 0 3 ul 25 ul 7 ine 10X 25 ul 0 3 ul 25 ul 8 T i 100X 25 ul 0 3 ul 25 ul 9 iG iD 25 ul 0 3 ul 25 ul 1X is a smallest appropriate amount of interfering peptide recombinant protein growth factor expected to modulate signaling pathway _1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support 888 503 3187 US 301 682 9200 23 Cignal Finder Reporter Arrays tube format Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 ul Opti MEM 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positive control Mix each tr
18. ic complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin Technical Support support SABiosciences com www SABiosciences com 24 Version 1 5 10 After 24 hours of transfection treat the cells as described in Table 12 with 1X 10X and 100X amount interfering peptide recombinant protein growth factor 1X is an smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 11 To study the effect of interfering peptide recombinant protein growth factor we recommend harvesting cells 6 hours or 18 hours after treatment to develop luciferase asSay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We found some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day befor
19. ic protocols within this user manual 2 Traditional Transfection Protocol Overview 2 DAY PROCEDURE Traditional Transfection Add Cells and Medium 009 ac 96 well Cell Culture Plate LEE DAY 1 Cignal Reporter and Add SureFECT Transfection me Test Nucleic Acids DAY 2 DAY 1 Trypsinize if necessary count and suspend cells to appropriate density Seed cells into multiwell plate s DAY 2 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or siRNAs Control transfections ii Cignal Reporter negative control for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs iv Cignal Negative Control negative control for test nucleic acid v Cignal Positive Control Dilute transfection reagent into appropriate medium If you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for transfection Add diluted transfection reagent to nucleic acid mixtures incubate at room temperature for 20 minutes Aliquot transfection complexes into wells containing overnight cell cultures Technical Support support SABiosciences com www SABioscienc
20. ignal Reporter Assays work well with transfection reagents from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV F This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 6 Guidelines for studying the effect of a peptide or recombinant protein Table 6 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Peptide or Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Recombinant DNA Diluent per well SureFECT Transfection per well Control Control Protein per well diluent hours per well per well per well per well 1 a a 25 ul 0 3 ul 25 ul 2 a i 1x 25 ul 0 3 ul 25 ul 3 ao k 10X 25 ul 0 3 ul 25 ul 4 Ve a 100X 25 ul 0 3 ul 25 ul 5 a i 25 ul 0 3 ul 25 ul 30
21. imited license conveyed with the purchase of this product are as follows Researchers may use this product in their own research and they may transfer derivatives to others for such research use provided that at the time of transfer a copy of this label license is given to the recipients and the recipients agree to be bound by the conditions of this label license Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the luciferase gene or Monster Green gene except that Researchers may 1 clone heterologous DNA sequences at either or both ends of said luciferase or Monster Green gene so as to create fused gene sequences provided that the coding sequence of the resulting luciferase or Monster Green gene has no more than four deoxynucleotides missing at the affected terminus when compared to the intact luciferase or Monster Green gene sequence and 2 insert and remove nucleic acid sequences in furtherance of splicing research predicated on the inactivation or reconstitution of the luminescent activity of the encoded luciferase In addition Researchers must do one of the following 1 use luminescent assay reagents purchased from Promega Corporation for all determinations of luminescence activity resulting from the research use of this product and its derivatives or 2 contact Promega Corporation to obtain a license for the use of the product and its derivatives No other use or transfer of this product o
22. ine HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 4 Guidelines for setting up co transfections of an expression vector and Cignal Reporter Assay Table 4 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol 35S 255l ese gs f Ssl vi s s bos l bes 5 Og 8 j ogS202S2 376E 2 87552 O 852 32 amp z52 FES Wi Ww z 1 a a 100 ng i 25 ul 0 3 ul 25 ul 2 he a 200 ng 25 ul 0 3 ul 25 ul 3 E a 100 ng it 25 ul 0 3 ul 25ul 32h 48h 4 46 Ki 200 ng 25 ul 0 3 ul 25 ul 5 HG a 100 ng pe 25 ul
23. l 100 ng Cignal negative control 200 ng experimental vector expressing gene of interest 1 ul 100 ng Cignal negative control 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal negative control 200 ng negative control expression vector 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the nine tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 4 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 COz Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is im
24. or reproduction of any constructs to modify kit components for resale or to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppel is granted NOTICE TO PURCHASER II The Dual Luciferase Reporter Assay and Monster Green Fluorescent Protein are trademarks of Promega Corporation Technical Support 888 503 3187 US 301 682 9200 27 Cignal Finder 10 Pathway Reporter Arrays tube format Part 1034A Version 1 5 1 10 2011 J SABiosciences A QIAGEN Company 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com
25. ormat 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of the diluted nucleic acids 1 1 ratio as detailed in Table 2 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs siIRNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing
26. portant to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct vector SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth Technical Support support SABiosciences com www SABiosciences com 18 Version 1 5 8 Incubate cells at 37 C in a 5 COs incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 11 To study the effect of the gene product we recommend harvesting cells 32 hours or 48 hours after transfection to perform the dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent
27. positive control construct can be monitored by fluorescence microscopy using an excitation filter of 470 20 nm 470 40 nm and an emission filter of 515 nm long pass Technical Support 888 503 3187 US 301 682 9200 7 Cignal Finder Reporter Arrays tube format Tandem repeats TATA box A of TREs y Firefly Luc B n CMV immediate early enhancer promoter I TATA box Firefly Luc CMV immediate early 1 enhancer promoter c y H Frey j ar CMV immediate early enhancer promoter Firefly Luc Figure 2 Schematic representation of constructs involved in the Cignal Reporter Assay A The inducible transcription factor responsive construct expressing firefly luciferase B The constitutively expressing Renilla luciferase construct C The non inducible firefly luciferase reporter construct D The constitutively expressing GFP construct and E The constitutively expressing firefly luciferase construct Technical Support support SABiosciences com www SABiosciences com 8 Version 1 5 Additional Materials Required Mammalian cell line cultured in the appropriate growth medium Cell culture medium and standard cell culture supplies 96 well tissue culture plates Multi channel pipettor and pipettor reservoirs Transfection reagent We recommend SureFECT SABiosciences Cat No SA 01 however other transfection reagents work equally well Polystyrene test tubes BD FALCON Cat 352099 Opti
28. r Assay The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 3 Guidelines for setting up co transfections of a shRNA vector and Cignal Reporter Assay Table 3 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive shRNA Control Nucleic per well SureFECT transfection per well Control Control per well shRNA Acid Diluent hours pe
29. r its derivatives is authorized without the express written consent of Promega Corporation including without limitation Commercial Use Commercial Use means any and all uses of this product and derivatives by a party for monetary or other consideration and may include but is not limited to use in 1 product manufacture and 2 to provide a service information or data and or resale of the product or its derivatives whether or not such product or derivatives are resold for use in research With respect to such Commercial Use or any diagnostic therapeutic or prophylactic uses please contact Promega Corporation for supply and licensing information If the purchaser is not willing to accept the conditions of this limited use statement SABiosciences is willing to accept the return of the unopened product and provide the purchaser with a full refund However in the event the product is opened then the purchaser agrees to be bound by the conditions of this limited use statement The above license relates to Promega Corporation patents and or patent applications on improvements to the luciferase and Monster Green gene NOTICE TO PURCHASER This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use The purchase of Cignal Reporter Assay kits 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 f
30. r well per well per well Diluent per well per well 100 ng 1 1 0 ul 200 ng 25 ul 0 3 pl 25 ul 100 ng 2 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 pl 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 200 ng sequence specific shRNA Control transfections 1 ul 100 ng Cignal reporter 200 ng negative control snRNA ul 100 ng Cignal negative control 200 ng sequence specific snRNA ul 100 ng Cignal negative control 200 ng negative control snRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 888 503 3187 US 15 Technical Support 301 682 9200 Cignal Finder Reporter Arrays tube format 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing
31. recommendations Important recommendations for best results A Perform all transfections in triplicate to minimize variability among treatment groups B Include positive and negative controls in each experiment to obtain reliable results C Use low passage cells that are actively growing and are greater than 90 viable for maximal transfection efficiencies D Do not add antibiotics to media during transfection as this may cause cell death E Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment F Serum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 5 in the assay medium during the experimental treatment to minimize these serum effects Technical Support support SABiosciences com www SABiosciences com 10 Version 1 5 B Generalized Transfection Protocols We recommend using reverse transfection protocols with the SureFECT transfection reagent throughout the Cignal Finder Reporter Arrays User Manual This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods However the Cignal Reporter Assays included in each Cignal Finder Array also work well with traditional forward transfection methods and transfection reagents from other vendors Below are general protocol overviews for the Cignal Reporter Assays using ei
32. riptional Response Element TRE Figure 2A This construct monitors both increases and decreases in the activity of a key transcription factor which is a downstream target of a specific signaling pathway The constitutively expressing Renilla construct encodes the Renilla luciferase reporter gene under the control of a CMV immediate early enhancer promoter Figure 2B and acts as an internal control for normalizing transfection efficiencies and monitoring cell viability It is also useful to confirm transfection and to verify active luciferase in the transfected culture 2 Negative control The negative control is a mixture of non inducible reporter construct and constitutively expressing Renilla luciferase construct 40 1 The non inducible reporter construct encodes firefly luciferase under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 2C The negative control is critical to identifying specific effects and determining background reporter activity 3 Positive control The positive control is a constitutively expressing GFP construct Figure 2D pre mixed with a constitutively expressing firefly luciferase construct Figure 2E and a constitutively expressing Renilla luciferase construct Figure 2B 40 1 1 The positive control is necessary for visual confirmation of transfection It is also useful for transfection optimization studies The expression of the GFP from the
33. room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 COz Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin Technical Support 888 503 3187 US 301 682 9200 21 Cignal Finder Reporter Arrays tube format 10 After 24 hours of transfection treat the cells as described in Table 5
34. s a final volume of 150 pl Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 11 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 16 Version 1 5 E Co transfection Protocol for Expression Vector Reporter Assay The following protocol is designed to reverse transfect an adherent cell l
35. s into your cell lines of interest The Cignal Finder Reporter Arrays are valuable tools for progressing from the identification of genes proteins or small molecules to understanding their function Each pathway focused dual luciferase reporter encodes for the mammalian codon optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly degrade the destabilized form of the firefly luciferase protein and hence the background luciferase activity noise level is greatly reduced Due to low background activity the magnitude of the response that can be measured signal to noise ratio as well as the speed of measuring changes in transcription are enhanced The Cignal dual luciferase reporter assays provide outstanding reproducibility sensitivity specificity and signal to noise ratio They are extremely useful assays for carrying out quantitative pathway regulation studies Benefits of Cignal Finder 10 Pathway Reporter Arrays e BIOLOGICAL PROCESS FOCUSED Profile the changes in the activities of ten signaling pathways relevant to a specific biological process e HIGH PERFORMANCE Dual luciferase assay provides high sensitivity specificity and reproducibility e FLEXIBILITY AND CONVENIENCE Utilize a straightforward traditional transfection or reverse transfection procedure with your favorite cell lines to rapidly generate valuable mechanism of action data Available Cignal Finder 10 Path
36. state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 19 Cignal Finder Reporter Arrays tube format F Transfection and Treatment Protocol for Reporter Assay Small Molecules Organic Compounds The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT SABiosciences Cat No SA 01 as a transfection reagent in 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors f you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general guideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 5 Guidelines for studying t
37. ther reverse or forward transfection approaches 1 Reverse Transfection Protocol Overview 1 DAY PROCEDURE Reverse Transfection 96 well Cell Culture Plate LEZLEY Add SureFECT Transfection Reagent Cignal Reporter and Test Nucleic Acids 9090 Seed Cells for Reverse Transfection OC DAY 1 LEE DAY 1 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or siRNAs Control transfections ii Cignal Reporter negative control for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs v Cignal Negative Control negative control for test nucleic acid Cignal Positive Control Dilute SureFECT into Opti MEM Add diluted SureFECT to nucleic acid mixtures incubate at room temperature for 20 minutes Trypsinize if necessary count and suspend cells to appropriate density Aliquot transfection complexes into wells Immediately seed cells to each well Technical Support 888 503 3187 US 301 682 9200 11 Cignal Finder Reporter Arrays tube format For detailed information on the transfection conditions and treatment of cultures post transfection refer to the application specif
38. way Reporter Arrays tube format Product Name Catalog Number Cignal Finder Cancer 10 Pathway Reporter Array CCA 001L Cignal Finder Development 10 Pathway Reporter Array CCA 003L Technical Support support SABiosciences com www SABiosciences com 4 Version 1 5 12 Tube Strip CAAA Add SureFECT Transfection Reagent Cignal Reporter amp Test Nucleic Acids Treat amp Analyze Phenotype with a Cell based Assay Oual luciferase Figure 1 Overview of Cignal Finder Reporter Array tube format Process Technical Support 888 503 3187 US 301 682 9200 5 Cignal Finder Reporter Arrays tube format ll Product Contents and Descriptions A Cignal Finder 10 Pathway Reporter Array Contents Table 1 Cignal Finder Reporter Array tube format Specifications Concentration Component Specification and total volume A mixture of an inducible transcription factor Each of the responsive firefly luciferase reporter and 100 ng ul 100 ul 10 Reporter constitutively expressing Renilla construct Assays 40 1 Negative A mixture of non inducible firefly luciferase control reporter and constitutively expressing 100 ng ul 100 ul Renilla construct 40 1 A mixture of a constitutively expressing GFP Positive construct constitutively expressing firefly 100 ng ul 100 yl control luciferase construct and constitutively expressing Renilla luciferase construct 40 1 1

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