Cignal Reporter Assay Kit User Manual
Contents
1. Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers 27 G Transfection and Treatment Protocol for Reporter Assay Peptide Recombinant Protein The following protocol is designed to reverse transfect adherent cell line HEK 293H using Attractene Transfection Reagent cat no 301005 in a 96 well plate format The Cignal reporter assay works well with transfection reagent from other vendors f you are using transfection reagent other than Attractene follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works 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 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 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 Attractene O2. 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 Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488 nm excitation and filters of 530 15 nm 530 30 nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20 nm and an emission filter of 515 nm long pass 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
3. before starting the experiment IMPORTANT 1 Do not add antibiotics to media durin causes cell death transfection as this Table 5 Guidelines for studying the 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 Attractene Opti MEM Time of Reporter Negative Positive Molecule DNA per well Attractene Transfection per well Control Control Organic diluent diluent hours per well Construct Compound per well per well per well per well 100 ng 1 1 0 ul 25 ul 0 6 ul 25 ul 100 ng a 2 1 0 ul 1X 25 ul 0 6 ul 25 ul 100 ng 3 1 0 ul 10X 25 ul 0 6 ul 25 ul 100 ng 4 1 0 ul 100X 25 ul 0 6 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 6 ul 25 ul 30 hor 42 h 100 ng 6 1 0 ul 1X 25 ul 0 6 ul 25 ul 100 ng 7 1 0 ul 10X 25 ul 0 6 ul 25 ul 100 ng 8 1 0 ul 100X 25 ul 0 6 ul 25 ul 100 ng 9 1 0 ul 25 ul 0 6 ul 25 ul 1X 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 w
4. spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488 nm excitation and filters of 530 15 nm 530 30 nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20 nm and an emission filter of 515 nm long pass 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 D Co transfection Protocol for shRNA Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using Attractene Transfection Reagent cat no 301005 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagent from other vendors f you are using transfection reagent other than Attractene Transfection Reagent follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are u
5. 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 transfection cocktail gently 2 Prepare an Attractene dilution for 9 tubes mentioned in step 1 by dispensing 5 4 ul of Attractene into 225 ul of Opti MEM serum free culture medium for every well dilute 0 6 ul of Attractene 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 Attractene to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted Attractene 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 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 4 x10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproduc
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7. 5 1 0 ul 100 ng ng 25 ul 0 6 ul 25 ul 100 ng 6 1 0 u 200 ng 25 ul 0 6 ul 25 ul 100 ng 100 7 4 0 ul 100 ng 25 ul 0 6 ul 25 ul 100 ng 8 4 0 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 9 1 0 ul 25 ul 0 6 ul 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 22 culture medium and Attractene to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 9 polystyrene tubes 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 ul 100 ng Cignal negative control 200 ng experimental vector expressing gene of inte
8. January 2011 Cignal Reporter Assay Handbook For cell based pathway activity assays QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Product Use Limitations Cignal Reporter Assay Kits are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease CONTENTS I Introduction IL Product Contents and Descriptions Ill Additional Materials Required IV Protocol A B H Before you begin Generalized Transfection Protocols Co transfection Protocol for siRNA Reporter Assay Co transfection Protocol for shRNA Reporter Assay Co transfection Protocol for Expression Vector Reporter Assay Transfection and Treatment Protocol for Reporter Assay Small Molecules Organic Compounds Transfection and Treatment Protocol for Reporter Assay Peptide Recombinant Protein Scaling u
9. P are slightly red shifted compared to other commercially available GFPs Peak excitation occurs at 505nm with a shoulder at 480nm peak emission occurs at 515nm Benefits of Cignal Reporter Assays e PERFORMANCE Both the dual luciferase and GFP reporter systems provide exceptional sensitivity reproducibility specificity and signal to noise ratio e VERSATILITY Can monitor signal transduction pathway activity utilizing the dual luciferase reporter system in an endpoint format assay or measure pathway activation dynamics on live cells using the GFP reporter system e CONVENIENCE Transfection ready constructs including positive and negative controls coupled with a transient reporter system enable rapid analysis of signal transduction pathway regulation How CIGNAL REPORTER LUC ASSAYS WORK Figure 1A Overview of Cignal Dual Luciferase Reporter Assays Process How CIGNAL REPORTER GFP ASSAYS WORK Figure 1B Overview of Cignal GFP Reporter Assays Process a Product Contents and Descriptions A Dual Luciferase Reporter Assay Kits 1 Kit Contents Table 1 Cignal Reporter Assay Kit Specifications Concentration Component Specification and total volume A mixture of an inducible transcription factor Reporter responsive firefly luciferase reporter and 100 ng ul 500 ul constitutively expressing Renilla construct 40 1 Negative A mixture of non inducible firefly luciferase control reporter and cons
10. TF4 ATF3 ATF2 CCS 8034L Androgen Androgen Receptor CCS 1019L Antioxidant Response Nrf2 amp Nrf1 CCS 5020L ATF6 ATF6 CCS 9031L C EBP C EBP CCS 001L CCS 1001G cAMP PKA CREB CCS 002L CCS 002G Cell Cycle E2F DP1 CCS 003L DNA Damage p53 CCS 004L Early Growth Response EGR1 CCS 8021L CCS 3021G ER Stress CBF NF Y YY1 CCS 2032L Estrogen Receptor Estrogen Receptor ER CCS 005L GATA GATA CCS 1035L Glucocorticoid Receptor Glucocorticoid Receptor GR CCS 006L Heat Shock Response HSF CCS 4023L Heavy Metal Response MTF1 CCS 5033L CCS 0033G Hedgehog Gli CCS 6030L Hepatocyte Nuclear Factor 4 HNF4 CCS 3039L Hypoxia HIF 1 CCS 007L Interferon Regulation IRF 1 CCS 7040L Interferon Type STAT1 STAT2 CCS 008L CCS 008G Interferon Gamma STAT1 STAT1 CCS 009L KLF4 KLF4 CCS 4036L CCS 9036G Liver X Receptor LXRa CCS 0041L 32 MAPK ERK Elk 1 SRF CCS 010L CCS 010G MAPK JNK AP 1 CCS 011L CCS 011G MEF2 MEF2 CCS 7024L c Myc Myc Max CCS 012L Nanog Nanog CCS 7037L NF B NFkB CCS 013L CCS 013G Notch RBP Jk CCS 014L CCS 1014G Oct4 Oct4 CCS 0025L Pax6 Pax6 CCS 3042L PISK AKT FOXO CCS 1022L CCS 6022G PKC Ca NFAT CCS 015L PPAR PPAR CCS 3026L Progesterone Progesterone Receptor CCS 6043L Retinoic Acid Receptor RAR CCS 016L Retinoid X Receptor RXR CCS 9044L Sox2 Sox2 CCS 0038L SP1 SP1 CCS 6027L CCS 1027G STAT3 STAT3 CCS 9028 TGFB SMAD2 SMAD3 SMAD4 CCS 017L CCS 017G Vitamin D VDR CCS 2029L Wn
11. 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 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 Attractene Opti MEM Time of Reporter Negative Positive siRNA Control Nucleic per well Attractene 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 6 ul 25 ul 100 ng 2 1 0 ul 2 pmol 25 ul 0 6 ul 25 ul 100 ng 3 1 0 ul 2 pmol 25 ul 0 6 ul 25 ul 48hor72h 100 ng 4 1 0 ul 2 pmol 25 ul 0 6 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 6 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 Attractene to co
12. c 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 Attractene into Opti MEM Add diluted Attractene 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 For detailed information on the transfection conditions and treatment of cultures post transfection refer to the application specific protocols within this user manual 2 Traditional Transfection Protocol Overview 2 DAY PROCEDURE Traditional Transfection Add Cells and Medium V 96 well Cell Culture Plate DAY 1 and Test Nucleic Acids Trypsinize if necessary count and suspend cells to appropriate density Seed cells into multiwell plate s Add Transfection Reagent v DAY 2 DAY 1 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 15 Experimental transfection i Cignal Reporter test
13. d via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488 nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20 nm and an emission filter of 515 nm long pass 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 30 H Scaling up transfection experiments To transfect cells in different tissue culture formats vary the amounts of constructs number of cells and volume of Attractene 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 diff
14. e If your stimulus is one of the very few reported activators of the CMV regulatory element we advise using an alternative reporter as an internal control e W Bruening B Giasson W Mushynski and H D Durham 1998 Nucleic Acids Research 26 2 486 489 Activation of stress activated MAP protein kinases up regulates expression of transgenes driven by the cytomegalovirus immediate early promoter e Madhu S Malo Moushumi Mozumder Alexander Chen Golam Mostafa Xiao Bo Zhang Richard A Hodin 2006 Analytical Biochemistry 350 307 309 pFRL7 An ideal vector for eukaryotic promoter analysis III 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 Attractene Transfection Reagent QIAGEN cat no 301005 however other transfection reagents work equally well Polystyrene test tubes BD FALCON Cat 352099 Opti MEM I 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 wh
15. eat 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 4 x10 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 shRNA Attractene complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 4 x10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct shRNA Attractene 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 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 deve
16. egrade 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 for carrying out endpoint pathway regulation assays The Cignal GFP reporter assays enable you to monitor the dynamics of pathway activation on living cells with single cell resolution The Cignal GFP reporter constructs utilize the Monster Green Fluorescent Protein reporter gene Monster GFP is encoded by an improved synthetic version of the green fluorescent protein gene This GFP expression cassette has been codon optimized to maximize mammalian cell expression and also utilizes an optimized Kozak sequence to increase translation efficiency The synthetic GFP is an ideal fluorescent reporter providing high level fluorescence and minimal cytotoxicity Moreover the synthetic GFP gene is resistant to photobleaching In addition most consensus sequences for transcription factor binding have been removed from the synthetic GFP gene in order to minimize aberrant transcription and improve the reliability of the GFP as an accurate reporter The spectral properties of the synthetic GF
17. ells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free 25 culture medium and Attractene to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 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 an Attractene dilution for 9 tubes mentioned in step 1 by dispensing 5 4 ul of Attractene into 225 ul of Opti MEM serum free culture medium for every well dilute 0 6 ul of Attractene 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 Attractene to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted Attractene into the positive control tube containing 25 ul of diluted con
18. en 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 FACS flow cytometer fluorescent microscope or fluorometer LV 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 Attractene Transfection Reagent cat no 301005 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 Ci
19. erent size culture vessels Type of Surface Starting Starting Starting Volume of Starting No Volume of siRNA shRNA Plate Area amount Volume of Volume of Cell of Adherent Opti MEM Vector or Gene cm per of Attractene Attractene Suspension Cells per Medium Expression well construct ul well ul well ul well Well ul Vector ng well per Well 96 well 0 3 100 0 6 0 3 100 40 000 2 X 25 2 pmol 200 ng 48 well 0 95 150 1 8 0 8 250 130 000 2X 50 5 pmol 500 ng 24 well 1 9 250 3 2 1 6 500 250 000 2X 50 10 pmol 750 ng 12 well 3 8 500 6 4 3 2 1000 500 000 2 X 100 20 pmol 1 5 ug 6 well 9 4 1000 16 0 8 0 2500 1500 000 2 X 250 50 pmol 4 0 ug 35 mm 8 0 1000 16 0 8 0 2500 1500 000 2 X 250 50 pmol 4 0 ug 6 60 mm 21 2000 36 0 18 0 5000 3 0 X 10 2 X 500 100 pmol 8 0 ug 6 100 mm 55 5000 90 0 45 0 15000 15 9 0 X 10 2 X 1500 300 pmol 25 ug ml a 2X means one volume of Opti MEM medium for diluting constructs and another volume of Opti MEM medium for diluting Attractene 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 3 Appendix Cignal Reporter Assay Kits Dual luciferase Cat No MGFP Pathway Transcription Factor Cat No Amino Acid Deprivation A
20. fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 4 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 siRNA Attractene complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 4 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs siRNA Attractene 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 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 Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The
21. gnal 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 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 t
22. his 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 B Generalized Transfection Protocols We recommend using reverse transfection protocols with the Attractene Transfection Reagent throughout the Cignal Reporter Assays User Manual This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods However Cignal Reporter Assays 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 either reverse or forward transfection approaches 1 Reverse Transfection Protocol Overview 1 DAY PROCEDURE Reverse Transfection well Cell Culture Plate SSZ2SSSSSSSSEE FEFFFEFFEFER LLII Add Transfection Reagent and Test Nucleic Acids I i 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 nuclei
23. hnical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN T Sample amp Assay Technologies UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999
24. ible 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 29 7 Add 100 ul of prepared cell suspension 4 x10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs Attractene 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 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 Expression of the Monster GFP reporter can be monitore
25. ion Reagent follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works 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 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 as E 5 2 S am o gt o 2 CHtew fe ee ES CeT v Oo BLI vz 0ov gt 00 ony 5 2to Ga wWwtes o wi o os TELE ELTEL es oss essgs g Soss FF Sess oes Fes 29552455 585285 58225 s 2525 Fs gas EBS Og 8 02080208 amp yoFs 29858 O Eyes H H858 FEE gt x lt lt i W W z 100 ng 100 1 1 0 ul 100 ng 25 ul 0 6 ul 25 ul 100 ng 2 10 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 100 i da 100 ng ng 25 ul 0 6 ul 25ul 32h 48h 100 ng 4 1 0 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 100
26. ively 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 B GFP Reporter Assay Kits 1 Kit Contents Table 2 Cignal GFP Reporter Assay Kit Specifications Concentration Component Specification and total volume An inducible transcription factor responsive Reporter GFP reporter 100 ng ul 500 ul Negative A GFP reporter construct in which GFP control expression is controlled by a minimal 100 ng ul 500 ul promoter Positive A constitutively expressing GFP construct control 100 ng ul 250 ul 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 2 Description Each Cignal GFP Reporter Assay Kit includes the following components 1 Reporter The inducible transcription factor responsive GFP reporter encodes the green fluorescent protein gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE Figure 3A 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 2 Negative control The
27. loping the assay Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially 20 available GFP reporters We recommend using the standard FACS settings of an argon laser 488 nm excitation and filters of 530 15 nm 530 30 nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20 nm and an emission filter of 515 nm long pass 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 21 E Co transfection Protocol for Expression Vector Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using Attractene Transfection Reagent cat no 301005 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagent from other vendors f you are using transfection reagent other than Attractene Transfect
28. mpensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM O to each of 5 polystyrene tubes 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 2 Prepare an Attractene dilution for 5 tubes mentioned in step 1 by dispensing 3 ul of Attractene into 125 ul of Opti MEM serum free culture medium for every well dilute 0 6 ul of Attractene 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 Attractene 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 17 humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of
29. negative control is a GFP reporter that encodes the green fluorescent protein under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 3B The negative control is critical to identifying pathway specific effects and determining background reporter activity 3 Positive control The positive control is a constitutively expressing GFP construct Figure 3C The positive control is necessary for visual confirmation of transfection It is also useful for transfection optimization studies Tandem repeats TATA box of TREs A TATA box Figure 3 Schematic representation of constructs involved in Cignal GFP Reporter Assay A The inducible transcription factor responsive reporter expressing GFP B The GFP reporter controlled by a minimal promoter negative control C The constitutively expressing GFP construct positive control IMPORTANT NOTE There are a few reports in the literature of the CMV regulatory element being activated by certain stimuli see below We recommend that you confirm that the stimulus used in each Cignal reporter assay does not induce the CMV regulatory element in order to confirm that the CMV Renilla construct is the appropriate normalization construct for your experiment This can be done empirically by testing the impact of your stimulus on the Cignal positive control reporters which are each under the control of the CMV enhancer promoter cassett
30. 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 Attractene Transfection Reagent into appropriate medium If you are using a transfection reagent other than Attractene Transfection Reagent 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 C Co transfection Protocol for siRNA Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using Attractene Transfection Reagent cat no 301005 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagents from other vendors f you are using a transfection reagent other than Attractene Transfection Reagent follow their manufacturer s protocol for optimizing transfection The Cignal Reporter Assay also works 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 guideline the optimal conditions
31. o useful to confirm transfection and to verify active luciferase in the transfected culture 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 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 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 Tandem repeats TATA box of TREs 4 A Firefly Luc TATA box v C Firefly Luc E eier 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 constitut
32. or research use only The terms of the limited 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 o
33. p Transfection Experiments Appendix Cignal Reporter Assay Kits and Controls Ordering information 12 13 13 14 16 22 25 28 31 32 34 I Introduction The Cignal Reporter Assays are designed for accurate sensitive and quantitative assessment of the activation of signal transduction pathways SABiosciences has developed a series of inducible reporter constructs that encode a reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of specific transcriptional response elements TRE Figures 1A and 1B Transcription factor activities can act as readouts for the intracellular status of many signal transduction pathways Our constructs are specifically engineered for measuring changes in activity both increases and decreases of these signaling pathways Each of the Cignal reporter assays is available in a dual luciferase format In addition six of the Cignal reporter assays are also available as GFP reporter constructs These include the CRE SRE AP 1 NFkB SMAD and TCF LEF Cignal Reporter Assays The Cignal reporter assays are valuable tools for deciphering gene function as well as determining the mechanism of action of proteins peptides ligands and small molecule compounds Each of the dual luciferase formatted reporters encodes for the mammalian codon optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly d
34. pti MEM Time of Reporter Negative Positive Recombinant DNA per well Attractene Transfection per well Control Control Protein Diluent diluent hours er well er well er well er well er well 1 E a 25 ul 0 6 ul 25 ul 2 ee i 1x 25 ul 0 6 ul 25 ul 3 ti 10x 25 ul 0 6 ul 25 ul 4 ao i 100x 25 ul 0 6 ul 25 ul 5 ap 25 ul 0 6 ul 25 ul 30 h or 42h 6 Gn T 1x 25 ul 0 6 ul 25 ul 7 io uD 10x 25 ul 0 6 ul 25 ul 8 is i 100x 25 ul 0 6 ul 25 ul 9 iG a 25 ul 0 6 ul 25 ul 4X 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 28 culture medium and Attractene to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 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
35. py or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20 nm and an emission filter of 515nm long pass 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 24 F Transfection and Treatment Protocol for Reporter Assay Small Molecules Organic Compounds The following protocol is designed to reverse transfect adherent cell line HEK 293H using Attractene Transfection Reagent cat no 301005 as a transfection reagent in 96 well plate format The Cignal reporter assay works well with transfection reagent from other vendors f you are using transfection reagent other than Attractene follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works 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
36. rest 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 an Attractene dilution for 9 tubes mentioned in step 1 by dispensing 5 4 ul of Attractene into 225 ul of Opti MEM serum free culture medium for every well dilute 0 6 ul of Attractene 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 Attractene 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 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 4 x10 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 c
37. sing plates or wells of different size adjust the components 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 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 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 Attractene Opti MEM Time of Reporter Negative Positive shRNA Control Nucleic per well Attractene transfection per well Control Control per well shRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 2 1 0 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 3 1 0 ul 200 ng 25 ul 0 6 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 200 ng 25 ul 0 6 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 6 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
38. structs 1 1 ratio as detailed in Table 5 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 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 4 x10 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 4 x10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs Attractene complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 26 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 10 After 24 hours of transfection treat the cells as described in Table 5 with
39. 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 Attractene 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 along with the following Experimental transfection 1 ul 100 ng Cignal reporter 200 ng sequence specific snRNA Control transfections 1 ul 100 ng Cignal reporter 200 ng negative control shRNA 19 1 ul 100 ng Cignal negative control 200 ng sequence specific shRNA 1 ul 100 ng Cignal negative control 200 ng negative control shRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare an Attractene dilution for 5 tubes mentioned in step 1 by dispensing 3 ul of Attractene into 125 ul of Opti MEM serum free culture medium for every well dilute 0 6 ul of Attractene 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 Attractene 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 tr
40. t TCF LEF CCS 018L CCS 018G Xenobiotic AhR CCS 2045L CCS 7045G 33 Cignal Reporter Assay Controls Control Construct Components Concentration And Volume Catalog Number A mixture of non inducible Cignal Negative Control firefly luciferase reporter 100 ng ul 500 pl CCS NCL LUC construct and constitutively expressing Renilla luciferase construct 40 1 A mixture of a constitutively Cignal Positive Control expressing GFP construct 100 ng ul 250 ul CCS PCL LUC constitutively expressing firefly luciferase construct and constitutively expressing Renilla luciferase element 40 1 1 Cignal Negative Control A GFP reporter construct in GFP which GFP expression is 100 ng ul 500 ul CCS NCG controlled by a minimal promoter Cignal Positive Control A constitutively expressing 100 ng ul 250 ul CCS PCG GFP GFP construct Ordering Information Product Contents Cat no Cignal Reporter Assay Assays in dual luciferase or GFP format Varies Kits For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor 34 Trademarks QIAGEN QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the Cignal Repor
41. ter Assay Kits to the following terms 1 The Cignal Reporter Assay Kits may be used solely in accordance with the Cignal Reporter Assay Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the Cignal Reporter Assay Handbook and additional protocols available at www qiagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated an BOOS The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qiagen come Firefly and or Renilla Luciferase and Monster Green Limited Use Label License READ THIS FIRST BEFORE OPENING PRODUCT F
42. ther use or transfer of this product or 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 United States Patent No 5 292 658 licensed from Millipore Corporation Dual Glo Dual Luciferase and Monster Green are trademarks of Promega Corporation Opti MEM is a registered trademark of Life Technologies 2011 QIAGEN all rights reserved O www qiagen com Australia Orders 1 800 243 8
43. titutively expressing 100 ng ul 500 ul Renilla construct 40 1 A mixture of a constitutively expressing GFP Positive construct constitutively expressing firefly 100 ng ul 250 ul control luciferase construct and constitutively expressing Renilla luciferase construct 40 1 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 2 Description Each Cignal Reporter Assay Kit includes the following components 1 Reporter The reporter is a mixture of 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 Transcriptional 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 als
44. ytometry device 23 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 4 x10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct vector Attractene 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 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 Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488 nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microsco
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