Attagraph User Manual
Contents
1. Biol 2002 Jan 31 35 1 47 53 Review No abstract available PMID 16248969 PubMed indexed for MEDLINE X Box Kakiuchi C Ishiwata M Hayashi A Kato T XBP1 induces WFS1 through an XBPRE Binding XBP1 Mr endoplasmic reticulum stress response element like motif in SH SY5Y cells J Protein 1 P Neurochem 2006 Apr 97 2 545 55 Epub 2006 Mar 15 PMID 16539657 PubMed in process2. 7 c B e gt gt 3 u e 9 u gt JJ NEL Factorial30 Transcription Reporter System User Manual For Factorial30 and Factorial30 XL Kits Ver 01 2008 ajta gene www attagene com Attagene Inc Table of Contents luigi m cht ti hatsct ted redid Mads attache tab 3 Principle of the FACTORIAL Technology 4 FACTORIALSO Product Line E I Neque 6 FACTORIAL30 Product Selection Guide 7 Ordering Information 8 FACTORIAL30 Operation 10 FACTORIAL Assay WorkfloW 12 Overview of the FACTORIAL 13 Experimental Protocol ea Oe ENS deo eA M dott 15 Transfection of Cells with FACTORIAL 15 IN Treatment Of Cells 5 17 RNA ISOLATION oes p repre onc etc tied Pen cte petri ede p tee preteen 17 IN DNase Treatment esi mn dien nin ae sonia boi ede 17 V Reverse TratnscPpllobt cuore beoe eot ue Ut hee up ale dU Ud 19 ARMES DD 20 VII Labeling FACTORIAL DNA by Primer Extension 23 VIII Digesting the Labeled FACTORIAL Primer Exte
3. protocol are performed using a homogeneous set of reagents in single reaction tube format providing highly uniform conditions for detecting multiple transcription factors adis Currently our FACTORIAL library comprises 30 individual TF responsive cis regulatory RTUs The promoter region of a typical RTU contains single or tandem repeats of a cis regulatory element that is specific for a particular TF or family of TFs sharing the same DNA binding specificity Response elements are positioned upstream of a minimal TATA like promoter that is common for all the TF responsive cis RTUs Transcription factors that can be evaluated using the FACTORIAL30 system are listed in Appendix B Many of these transcription factors represent signaling pathways with well established biological roles controlling cell proliferation stress responses inflammation drug metabolism and differentiation Each individual FACTORIAL30 RTU was functionally validated We performed a number of transient reporter assays in which we assessed the RTUs responsiveness to model stimuli e g Il 18 and TGF were used to evaluate the responsiveness of NF kB and TGFB RTUs respectively Additionally the performance of each cis RTU was tested by co transfection with expression vectors encoding corresponding transcription factors Inducibility of the FACTORIAL RTUs was assessed by quantitative RT PCR and compared to that produced by conventional luciferase or phosphat
4. Manual for further analysis of FACTORIAL data 26 Frequently Asked Questions FAQ Q What is the advantage of using FACTORIAL system over other TF profiling methods currently available Currently there are 2 types of assays of this kind available 1 DNA binding assays which allow measurement of a transcription factor s DNA binding activity but not its true activity 2 Reporter gene types of assays which allow measurement of TF activity but do not afford high content screening The FACTORIAL assay is a high content assay that allows you to measure true TF activity as opposed to DNA binding activity alone How many TFs can be studied simultaneously using FACTORIAL assay A Attagene s FACTORIAL30 Kit allows you to analyze the activity of 30 transcription factors in one homogenous assay What types of cells are suitable for the FACTORIAL assay A To be able to use the FACTORIAL assay with your cells of interest you must first make sure that your cells are transfectable You ll also need to optimize the transfection efficiency so that you have at least 10 000 cells transfected with the FACTORIAL reagent How can 1 determine which FACTORIAL version will give the best profile with my cell line At present there are 6 FACTORIAL versions available for researchers and those 6 versions were tested in 23 cell lines Please refer to Table 2 to see if your cell line of interest has
5. Optional Step To assess the degradation of the RNA run a sample about 1 ug from the DNase treatment on a 1X TAE or 1X TBE gel of 1 agarose 0 5 ug ml EtBr It is not necessary to run a formaldehyde gel for this analysis After running the gel the 18s and 28s ribosomal RNA bands should be sharp and the 28s band should be twice as 18 intense as the 18s band Alternatively integrity of your RNA samples be assessed on bioanalyzer RNA is subject to degradation by both enzymatic and chemical processes so extreme care should be taken when isolating storing or using RNA The RNA from this step may be stored at 80 C until you are ready to proceed V Reverse Transcription The DNase treated RNA is now ready to be reverse transcribed into cDNA which will then be amplified via PCR General notes e sure to use RNase free tips tubes and solutions for this section of the protocol Gloves should be worn at all times to minimize RNase contamination e Two reactions RT and a RT control should be assembled for each RNA sample isolated in Section Ill The RT negative control reaction is recommended for proper identification of the correct size PCR product in RT reaction see below Protocol Note Amounts in the following protocol are given per single reaction We advise preparing a Master Mix for all the samples plus one extra reaction 1 Assemble the following RT primer annealing reaction 9 ul of
6. buffer 250 pl 17 Taq DNA Polymerase 25 ul 18 RNase free water 3 ml Additional Reagents Required not supplied by ATTAGENE Transfection reagent RNA isolation reagents DNA Mass Ladder such as Low DNA Mass Ladder Invitrogen cat 10068 013 Performa DTR gel filtration cartridges Edge Biosystems cat 42453 or Qiagen PCR Purification Kit cat 28104 Formamide Hi Di Applied Biosystems cat 4311320 10 Instruments required Thermocycler Bench centrifuge Capillary electrophoresis instrument ABI sequencer Fragment Analyzer ATTAGRAPH software available from www attagene com ABI sequencers Fragment Analyzers are usually available from DNA sequencing facility If you do not have the instrument available you can submit and send your processed samples to us See detailed instructions on page 26 11 FACTORIAL Assay Workflow FACTORIAL Assay Step Estimated Time Transfect cells to be evaluated with FACTORIAL Reporter Library 24 hrs Treat cells as desired Variable Isolate total RNA or mRNA 30 minutes i Treat RNA with DNase 1 hour Reverse id id RNA into cDNA 2 hours Amplify FACTORIAL cDNA by PCR 2 hours Label PCR product by primer extension 15 minutes Digest labeled DNA with Hpa 2 4 hours Purify FACTORIAL DNA 1 5 hours Run capillary electrophoresis 1 5 hours Analyze data using ATTAGRAPH Software Variable 212 Overview of the FACTORIAL Assay The FACTORIAL
7. oxygen homeostasis Kaelin WG Proline hydroxylation and gene expression Annu Rev Biochem 2005 74 115 28 Review PMID 15952883 PubMed indexed for MEDLINE Nagarsekar A Hasday JD Singh IS CXC chemokines A new family of heat shock HSE HSF1 2 geldanamycin stress proteins Immunol Invest 2005 34 3 381 98 Review PMID 16136787 PubMed in eat shock heat shock process IRE 1 3 immune responses host defense Battistini A Marsili G Sgarbanti M Ensoli B Hiscott J IRF regulation of HIV 1 long ISRE ISRE family interferon cytokine signaling cell growth terminal repeat activity J Interferon Cytokine Res 2002 Jan 22 1 27 37 Review Y regulation PMID 11846973 PubMed indexed for MEDLINE TATA Minimal promoter Sala A B MYB a transcription factor implicated in regulating cell cycle apoptosis and MYBRE MYB MYB stem cell formation differentiation cancer Eur J Cancer 2005 Nov 41 16 2479 84 Epub 2005 Sep 29 Review PMID 16198555 PubMed indexed for MEDLINE NF Kb RelA immune response inflammatory Dolcet X Llobet D Pallares J Matias Guiu X NF kB in development and progression NFKBRE Family TNF stress proliferative and apoptotic of human cancer Virchows Arch 2005 May 446 5 475 82 Epub 2005 Apr 27 Review responses PMID 15856292 PubMed indexed for MEDLINE NRF1 NFE2L1 mitochondrial genesis Jaiswal AK Nrf2 signaling in coordinated activation of antioxidant gene NRF1RE NRF 1 antioxidative respons
8. the RNA from Section IV 3 ul RNase free water 1 ul dNTPs 10 mM each 1 ul RT Primer 0 5 ug ul Final reaction volume 14 ul 2 Incubate at 70 C for 3 minutes then cool the reaction at room temperature for 5 minutes This step can be performed in a thermocycler 3 Add the following components to each of your annealing reactions 2 ul 10X RT Buffer 0 4 ul MgCl 50 mM 2 6 ul RNase free water 1 ul M MuLV Reverse Transcriptase 200 units ul 19 Final reaction volume 20 ul per reaction Note Extra is added to compensate for excess of EDTA 4 Incubate for 1 5 hours at 37 C then for 15 minutes at 70 C Reminder A negative control reaction RT for each RNA sample should be assembled with the same reagents as the RT reaction except there is no reverse transcription enzyme present Both reactions should be incubated identically The RT products can be stored at 20 C until you are ready to proceed VI PCR Amplification After reverse transcription the FACTORIAL cDNA must be amplified by PCR Notes We recommend that you reserve one set of pipettes for PCR set up purposes only and perform the PCR set up in a specially designated area to avoid carry over problems e Since PCR can amplify small amounts of DNA take care to limit the amount of contamination of the PCR reaction Use aerosol tips and gloves when assembling the PCR reaction Dueto the sensitivity of the technology to the PCR ampli
9. 5 ug 40 80 000 12 well dish 0 5 ug 50 60 000 24 well dish 0 25 ug 70 40 000 24 well dish 0 25ug 90 20 000 48 well dish 0 1 ug Note Depending on the tissue culture cells used you may wish to change the plate format suggested FACTORIAL detection has been successfully completed in the following cells using the FuGene 6 Roche transfection protocol outlined below C3H 10T1 2 U 87 MG NIH 3T3 COS 7 HepG2 INS 1 NHDF 293tk Raw264 7 K562 HeLa MCF 7 SW480 HCT116 SH SY5Y ZR 75 1 CHO K1 3T3 L1 MDCK C2C12 Caco 2 VERO FuGene 6 Roche Transfection Protocol 1 2 RO Plate healthy cells more than 24 hours prior to transfection Add 3 ul of FuGene to 97 ul of Optimem being sure to avoid touching the pipet tip to the side of the tube because the FuGene will stick to plastic Incubate for 5 minutes at room temperature Add 1 ul of FACTORIAL Reagent and co transfected plasmid if applicable to 50 ul of Optimem and mix well by tapping tube Mix the FuGene Optinem and DNA Optimem into a single tube and incubate at room temperature for 15 minutes Remove media from cells and replace with 900 ul of fresh media in each dish Add Optimem DNA FuGene mixture to cells Incubate overnight at 37 C Remove the media the next day rinse once with fresh media and put fresh media on the cells 10 Treat cells according to your experimental design
10. CTORIAL libraries Table 2 Cell types compatible with each FACTORIAL30 configuration FACTORIAL30 configuration Cell types tested FACTORIAL30 A 3T3 CHO K1 U87MG HCT116 COS 7 ZR 75 1 SH Sy5y FACTORIAL30 B 3T3 L1 C3H 10T 1 2 MDCK MCF 7 C2C12 K 562 RAW264 7 INS 1 FACTORIAL30 C HeLa FACTORIAL30 D Caco 2 HepG2 SW480 VERO FACTORIAL30 E NHDF FACTORIAL30 F HEK 293 Ordering Information Table 3 FACTORIAL30 kit names and catalog numbers Kit name Catalog number FACTORIAL 30 A FPS 03020A FACTORIAL 30 B FPS 03020B FACTORIAL 30 C FPS 03020C FACTORIAL 30 D FPS 03020D FACTORIAL 30 E FPS 03020E FACTORIAL 30 F FPS 03020F FACTORIAL 30 Explorer FPS 03020 Explorer FACTORIAL 30 A XL FPS 03020A XL FACTORIAL 30 B XL FPS 03020B XL FACTORIAL 30 C XL FPS 03020C XL FACTORIAL 30 D XL FPS 03020D XL FACTORIAL 30 E XL FPS 03020E XL FACTORIAL 30 F XL FPS 03020F XL FACTORIAL 30 XL Explorer FPS 03020 XL Explorer FACTORIAL30 TF Profiling Systems Each FACTORIAL30 TF profiling system is supplied with one particular configuration of the FACTORIAL30 Reporter Library The package also includes a set of essential FACTORIAL specific reagents RT and PCR primers labeling primer Hpa l Proteinase solution CE calibration reagent and CE capillary molecular weight standards
11. Each system is sufficient to perform 20 TF profiling reactions FACTORIAL30XL TF Profiling Systems These packages contain all the components of the corresponding FACTORIAL30 system but they also include additional reagents that are required for performing the detection steps of the assay including DNase enzyme RT and PCR enzymes with corresponding reaction buffers and dNTPs Alternatively these additional reagents can be obtained from other suppliers Both FACTORIAL30 and FACTORIAL30XL TF profiling systems are also available in the Explorer format which includes aliquots of all six configurations of the FACTORIAL30 reporter library ATTAGRAPH Reader Software ATTAGRAPH Reader Software is required for analysis of data generated using the FACTORIAL30 M system and is available for our customers thru Attagene s website at www attagene com How to order Orders to be shipped within the United States can be placed 24 hours a day by fax or e mail Customer Service Representatives are available between 9 00 am and 5 00 pm Eastern Time Monday through Friday to take your order In most cases orders are shipped the same day No minimum order quantity is required To place an order Telephone Toll free 1 888 721 2121 or 919 313 0473 Fax 919 313 0172 E mail orders attagene com Online http www attagene com Mail ATTAGENE PO Box 12054 RTP NC 27709 USA FACTORIAL 30 OPERATION MANUAL Please read the en
12. Other transfection protocols that have been successfully used with the FACTORIAL analysis include Qiagen s SuperFect Invitrogen s Lipofectamine 2000 Qbiogene s jetPEI and electroporation 16 ll Treatment of Cells With the FACTORIAL system time points may need to be taken sooner than in other assays because the method of detection is at the level of RNA rather than protein Because there are myriad potential experimental treatments we suggest that you use the compounds concentration and treatment times that you deem appropriate for the treatment and cell types to be utilized Ill RNA Isolation After transfecting your tissue culture cells with the FACTORIAL Reagent and treating them as desired you will need to isolate RNA to assess the level of transcriptional activity within the cell as identified by the FACTORIAL Reporters Note Be sure to use RNase free tips tubes and solutions for this section of the protocol Gloves should be worn at all times to minimize RNase contamination The best method for RNA isolation depends upon the scale of the RNA isolation and your personal preferences Multiple column based RNA isolation technologies used according to the manufacturers specifications have proven compatible with the FACTORIAL system Alternative methods of RNA isolation have also been used with good results The most important aspect of the isolation procedure is to purify intact RNA that is sui
13. S ETS famil ELK1 MAP kinase mediated signaling Seth A Watson DK ETS transcription factors and their emerging roles in human y PMA differentiation cancer Eur J Cancer 2005 Nov 41 16 2462 78 Epub 2005 Oct6 Review PMID 16213704 PubMed indexed for MEDLINE maintenance ot alucos and fond Wang H Wollheim CB Does chasing selected Fox to the nucleus prevent diabetes FOXARE FoxA family FoxA2 h guco Trends Mol Med 2005 Jun 11 6 262 5 Review PMID 15949766 PubMed indexed omeostasis for MEDLINE 32 FOXORE FoxO family FoxO 1 3 cell cycle arrest stress resistance or Greer EL Brunet A Related Articles Links FOXO transcription factors at the interface between longevity and tumor suppression Oncogene 2005 Nov 14 24 50 7410 25 apoptosis Review PMID 16288288 PubMed indexed for MEDLINE Bresnick EH Martowicz ML Pal S Johnson KD Developmental control via GATA GATARE GATA family GATA 2 4 differentiation factor interplay at chromatin domains J Cell Physiol 2005 Oct 205 1 1 9 Review PMID 15887235 PubMed indexed for MEDLINE GR NR3C1 Geserick C Meyer HA Haendler B The role of DNA response elements as allosteric GRE GR dexamethazone differentiation inflammation modulators of steroid receptor function Mol Cell Endocrinol 2005 May 31 236 1 2 1 7 Review PMID 15876478 PubMed indexed for MEDLINE HRE HIF1alpha HIF1a angiogenesis hypoxia
14. already been tested by Attagene If your cell line is not listed in that table we offer the Explorer version of the FACTORIAL kit The Explorer Kit contains trial amounts of all6 FACTORIAL versions so that you can determine which version works best in your cells If none of the current versions produces a satisfactory profile in your cells we can customize a FACTORIAL version for your cells of interest For more information and pricing please contact Attagene s Technical Support by phone at 1 888 721 2121 or by e mail at info attagene com What if our sequence facility does not provide CE analysis service A You may submit and send your samples to Attagene s service facility Please follow the instructions on sample preparation and shipping listed in Section X Your samples will be analyzed and results will be sent to you via e mail 27 Troubleshooting Guide Problem 1 Insufficient or no PCR product detected from the reverse transcription reaction Cause The transfection was unsuccessful Solution Make sure that your cells are maintained under the best conditions and that they look healthy before and after transfection If cells show poor viability after transfection then your current transfection protocol may be incompatible with the cells Make sure that you have optimized your transfection conditions so that cell viability is not affected and that you have a sufficient number of transfected cells to work w
15. ase based reporter gene constructs We found that RNA based detection provided better responsiveness than conventional enzymatic based assays FACTORIAL30 Product Line All our current products feature the FACTORIAL30 TF profiling library Table 1 lists the TFs and cis regulatory elements that can be evaluated using the FACTORIAL30 library Table 1 FACTORIAL30 cis regulatory elements p53 FOXA 1 For more information on each TF please see Appendix We offer a number of FACTORIAL30 products listed in the section below FACTORIAL30 Product Selection Guide Since activities of individual TFs may vary substantially in different cell types we provide six different configurations or versions of the FACTORIAL30 library each assigned a set of particular cell types The table below will help you to determine which FACTORIAL30 configuration will best suit the cell line of your choice If you do not see the name of your cell line in the table we recommend the FACTORIAL30 Explorer Kit which contains trial samples of all six FACTORIAL30 configurations All configurations contain the same set of cis regulatory elements but differ in the amount of individual reporters To accommodate researchers with specific needs we can also create a unique custom made FACTORIAL30 configuration that will be specific for your cells of interest Please contact us for more information and pricing on custom made FA
16. assay workflow is illustrated on page 12 Here we briefly describe each step of the assay For complete details about each step of the FACTORIAL detection protocol please see the corresponding parts of this manual 1 Plating of cells and introduction of the FACTORIAL Reporter Library into cells We have successfully performed the FACTORIAL assay with a variety of different cell types Both primary cells and stable cell lines can be used The plating format depends on your choice of cells transfectability of the cells and desired experimental throughput Highly transfectable cells gt 50 of transfection efficacy can be reliably assayed in a 96 well format For most stable cell lines we use a 12 well format Using cells that are healthy and plating them at a consistent density are important factors that dramatically improve the reproducibility of the FACTORIAL assay The FACTORIAL Reporter Library is usually introduced into cells via a transient transfection protocol Your choice of transfection technique depends on your choice of cell type A variety of available transfection reagents as well as electroporation can be used It is important that the efficacy of transfection is sufficient to produce at least 10 000 transfected cells per tissue culture sample This ensures efficient and reproducible detection of the FACTORIAL Reporter RNAs ll Treatment of cells Starting the next day after transfection the cells can be treate
17. b 2008 Feb 24 Principle of the FACTORIAL Technology The key to the FACTORIAL technology Figure 1 is a library of uniformly constructed Reporter Transcription Units RTUs Each FACTORIAL RTU is made in a common plasmid backbone and contains a unique TF inducible promoter region fused to a transcribed reporter sequence When co introduced into a cell of interest the RTUs produce reporter RNAs in amounts commensurable with the activities of the corresponding TFs present in a cell To provide equal detection opportunities for different transcription factors all FACTORIAL RTUs are supplied with essentially identical reporter sequences To distinguish reporter sequences produced by different RTUs each sequence is provided with a short processing tag restriction cleavage site the position of which varies among the RTUs Thus reporter sequences can be discriminated upon cleavage with the corresponding processing enzyme The cleaved reporter species are separated by high resolution capillary electrophoresis CE and quantified The CE is performed by standard sequencing instrument that is run in Fragment Analysis mode TFA Hpal Me sites Library of Reporter me Constructs RTUs U Transcription 0 RT PCR I amplification Labeling and Digest with Hpal __ y U a Separation and os TFA detection x TER Figure 1 A key feature of the FACTORIAL technology is that all steps of the detection
18. ble as peaks of fluorescence migrating according to the position of the processing tag in the corresponding FACTORIAL RTUs XI Data analysis and storage To extract the transcription factor activity profile the raw data acquired during capillary electrophoresis have to be analyzed using the provided ATTAGRAPH Reader Software available on our website at www attagene com 4 Experimental Protocol I Transfection of cells with FACTORIAL Reagent The first step of the FACTORIAL analysis requires introducing the FACTORIAL Reagent into tissue culture cells We suggest that you repeat each control and experimental condition at least three times to verify the reproducibility of the experiment If an additional plasmid e g an empty vector or a vector containing your gene of interest is to be co transfected with the FACTORIAL Reagent we suggest at least a 1 10 ug ratio of vector to FACTORIAL Reagent For example do not combine more than 0 1 of the vector with 1 ug 1 ul of the FACTORIAL Reagent The FACTORIAL assay utilizes a standard transfection protocol for introducing the plasmid DNA into tissue culture cells We recommend that the transfection protocol produce gt 5 transfection efficiency of the FACTORIAL reagent into the cells being treated General transfection considerations apply to this procedure cells should be healthy proliferating well and plated at a consistent density to minimize va
19. d according to your experimental design For example cells can be exposed to a variety of chemical or biological compounds for different intervals of time We usually allow for three replicas for each treatment condition and we always include appropriate positive and negative controls lll Isolation of total RNA Following the experimental treatment total RNA is extracted from the cells To this end commercially available column based RNA purification systems can be used Depending on the throughput of your assay other RNA isolation methods i e Trizol based extraction can also be used RNA samples can be stored frozen at 70 C IV Treatment with DNase This step is required to remove all traces of FACTORIAL plasmid DNA from the RNA samples The treatment should be sufficient to prevent any significant PCR amplification in no RT control samples RNA samples treated with DNase can be stored frozen at 70 C V Reverse transcription The DNase treated RNA samples are reverse transcribed to produce the FACTORIAL Reporter cDNAs This step is performed using standard reverse transcription reagents FACTORIAL cDNA samples can be stored frozen at 70 C VI PCR To amplify the FACTORIAL Reporter cDNAs this step is performed using FACTORIAL M specific primers The PCR reactions are carried out using a standard thermocycler The minimal required number of PCR cycles may vary depending on your cell type and tissue cult
20. e control PCR with the FACTORIAL DNA provided with the kit Dilute a small aliquot of that DNA 10 100 pg ul and use 1 ul of the dilution as a template for PCR You should observe a band of approximately 850 bp in length If this band is absent and you are certain the reaction setup and protocol were performed correctly your PCR machine may need to be calibrated Cause Insufficient PCR product was detected Solution Increase the number of PCR cycles You can increase the cycle number up to 35 cycles Beyond 35 cycles you will see an increase in product in the RT control It is possible that your transfection was not adequate to introduce enough of the FACTORIAL reagent into your cells 28 Problem 2 Cells show poor viability after transfection Cause Cells were not healthy before transfection Solution Make sure you use proper tissue culture technique and media for your cells Cause Transfection parameters are incompatible with your cell type Solution Optimize transfection per manufacturer s suggestions Switch to a new transfec tion reagent Problem 3 RNA appears degraded or is absent from the gel Cause RNA is very susceptible to degradation RNases may have degraded the RNA Solutions Repeat the experiment and make sure that you follow all procedures recommended by the manufacturer of the RNA isolation kit It is possible that your tubes or RNase free water have become contaminated with RNases Mak
21. e sure that you wear gloves at all times during the isolation procedure e Make sure you have rinsed your cells after the transfection to remove any excess transfection reagent e Make sure you have added the EDTA after the DNase treatment to inhibit chemical degradation of the RNA Problem 4 Too much PCR product detected in the RT control reaction Cause The DNase reaction was insufficient to eliminate enough of the DNA from the isolated RNA Solution Repeat the DNase treatment step on the RNA and continue with the rest of the protocol Cause Contamination of your RT or PCR reagents Solution Run a PCR reaction control using water as your template If you are still getting a PCR product of about 850 nucleotides then you have contamination of at least one of your PCR reagents Procure new PCR reagents and proceed with the protocol after DNase treatment of the RNA For problems arising during CE data analysis please refer to our ATTAGRAPH software manual available from our website at www attagene com 29 Trademarks and Patent information Components of the FACTORIAL30 kit are covered by patents and patent applications filed in the USA and world wide by Attagene Inc Factorial Factorial30 ATTAGRAPH are trademarks of Attagene Inc See ATTAGRAPH software manual for additional end user limitations End user license agreement Attagene Inc grants to the purchaser of any FACTORIAL30 products limi
22. es expression Free Radic Biol Med 2004 May 15 36 10 1199 207 Review PMID 15110384 PubMed indexed for MEDLINE oxidative stress responses Jaiswal AK Nrf2 signaling in coordinated activation of antioxidant gene AME NIS NBN eee expression Free Radic Biol Med 2004 May 15 36 10 1199 207 Review PMID 15110384 PubMed indexed for MEDLINE CNS development Phillips K Luisi B The virtuoso of versatility POU proteins that flex to fit J Mol Biol OCTRE OCT family OCT1 POU2F1 Z 2000 Oct 6 302 5 1023 39 Review PMID 11183772 PubMed indexed for general housekeeping MEDLINE P53 TP53 stress response Johnson J Lagowski J Sundberg A Kulesz Martin M P53 family activities in P53RE p53 tumor suppression development and cancer relationship to melanocyte and keratinocyte carcinogenesis J DNA Damage DNA damage Invest Dermatol 2005 Nov 125 5 857 64 Review No abstract available PMID 16297181 PubMed indexed for MEDLINE 33 central nervous system PAXRE PAX family Pax6 beta cell development differentiation Manuel M Price DJ Role of Pax6 in forebrain regionalization Brain Res Bull 2005 Sep i 15 66 4 6 387 93 Epub 2005 Feb 24 Review Tan NS Michalik L Desvergne B Wahli W Multiple expression control mechanisms of PPRE PPAR family PPAR a 8 y metabolism including fatty acid peroxisome proliferator activated receptors and their target genes J Steroid Biochem GW 742 oxidation and lipogenesi
23. fication step it may be necessary to optimize the PCR conditions for your specific PCR machine Please see the Troubleshooting Guide if this is required Use as templates both the Reverse Transcription RT and the Reverse Transcription RT negative control from Section V Protocol Note Amounts in the following protocol are given per single reaction We advise preparing a Master Mix for all the samples plus one extra reaction 1 Assemble PCR reactions at room temperature 18 25 ul water 2 5 ul 10X PCR buffer 0 5 ul dNTP mix 1 0 FACTORIAL specific PCR Primer Mix 0 25 ul Taq DNA Polymerase 20 2 In thin walled tubes that fit your thermal cycler add 22 5 ul of the Master Mix to 2 5 ul of reverse transcription reaction either RT or RT from Section V The final reaction volume in each tube should be 25 ul Note To test if your PCR reagents have become contaminated with FACTORIAL DNA assemble at least one PCR reaction with just water as a template 3 Perform PCR using the conditions outlined as follows PCR conditions Please note that the denaturation temperature used for the FACTORIAL Assay PCR is 95 C instead of the more commonly used 94 C PCR Cycles The number of cycles used for the PCR depends on your particular experimental conditions We recommend using no more than 32 cycles to start the optimization procedure 4 To assess the PCR products run a 1X TAE or 1X TBE gel w
24. h dNTP 0 5 ul FACTORIAL specific Labeling Primer 0 25 ul PCR enzyme 3 In thin walled tubes that fit your thermal cycler add 22 5 ul of the Master Mix to 2 5 microliters of the PCR reaction from Section VI 125 to 50 ng of PCR product The final reaction volume in each tube should be 25 yl 4 In a thermal cycler run the following primer extension reaction 94 C for 3 minutes 95 for 20 seconds 68 C for 20 seconds 72 for 6 minutes You may store the labeled FACTORIAL primer extension product at 20 C away from light until you are ready to proceed gu VIII Digesting the Labeled FACTORIAL Primer Extension Product After the FACTORIAL reporter cDNAs have been labeled they should be digested The digesting step will allow the capillary electrophoresis to resolve the reporter cDNAs produced by each specific FACTORIAL reporter Note The primer extension product has been labeled with a light sensitive fluorophore and should be kept from being exposed to light as much as possible Protocol 1 Add 1 ul of Hpa I restriction enzyme directly to the 25 ul labeling reaction from Section VII 2 Mix the samples and centrifuge to collect all of the liquid into the bottom of the tube 3 Incubate for 2 4 hours at 37 C in the dark and proceed immediately to the next step Section IX IX Proteinase K Treatment The labeled FACTORIAL samples must be purified prior to capillary electrophoresis The proteinase K t
25. ith at least 10 000 cells per sample We strongly recommend that you include a transfection control such as a reporter plasmid containing or eGFP to assess the percentage of transfected cells Cause The RNA was degraded either during the RNA isolation step the DNase procedure or the reverse transcription reaction Solution Take an aliquot of the DNase treated RNA and run it on a 1 agarose gel If the 18s and 28s RNA bands are intact and do not appear smeared on the gel then the RNA is intact and the RT or PCR step was not completed properly Repeat the experiment from the transfection step forward taking care to minimize RNase activity in your sample Cause The reverse transcription reaction was unsuccessful Solution The RNA may have degraded during the procedure or the reverse transcription may have been incomplete If the RNA is still intact from the DNase treatment and a second attempt at PCR was unsuccessful treat another aliquot of RNA with DNase then repeat both the reverse transcription reaction and the PCR protocol To make sure that the reverse transcription reaction was successful you can run your PCR on an aliquot of your RT reaction using primers commonly for ubiquitously expressed genes such as actin or HPRT Cause The PCR reaction was incomplete Solution Repeat the PCR step making sure that all of the components have been added and you are using the correct PCR protocol We suggest that you run a positiv
26. ith 1 agarose 0 5 ug ml EtBr Load 5 ul each of the RT and RT samples in separate lanes on the gel Load 5 ul of the DNA Mass Ladder alongside the samples to estimate the size and amount of DNA obtained from the PCR reactions Expected results e The PCR products in RT reactions should be 680 bp in length Although the RT samples should theoretically generate no bands some PCR product is occasionally detected DNA detected in the RT sample will have product size different from that of RT PCR product see Figure 2 Figure 3 shows an example of the acceptable amount range of PCR products that can be used for the labeling reaction Here 5 ul of the DNA Mass Ladder and 5 ul of the PCR product were loaded on an agarose gel 91 Figure 2 Electrophoretic analysis of PCR products on 1 agarose gel LANE 1 2 3 4 5 6 7 8 9 830bp RT 680bp RT Lane 1 5 pl of DNA Mass Ladder Invitrogen cat 10068 013 Lane 2 Acceptable amount of PCR product Lane 3 Acceptable amount of PCR product Lane 4 Acceptable amount of PCR product Lane 5 Too little PCR product Lane 6 Too little PCR product Lane 7 Too little PCR product Lane 8 Product of RT reaction Lane 9 100 bp DNA Ladder f no PCR product is visible in the RT sample see the Troubleshooting guide for suggestions on how to proceed The intensity of the RT PCR product band should be similar or exceeding the inten
27. ng their attention to Appendix A Ensure that the size standards supplied with this kit are the only molecular weight standards included in your sample If other standards are used it will not be possible to analyze the resulting data If two or more standards are mixed with your sample the analysis cannot be completed To obtain good capillary electrophoresis runs the manufacturers protocol for fragment analysis should be completed The fragments will range from about 90 nucleotides to 700 nucleotides in length See Appendix A for settings for an ABI 3130xl Genetic Analyzer machine Every time an analysis is run it is necessary to include four samples with calibration standards These standards supplied with the FACTORIAL kit allow the ATTAGRAPH Software to correctly interpret the data generated by the sequencing machine These standards should be treated as your other samples are treated for electrophoresis after Section IX Sample electrophoresis protocol for ABI sequencers 1 2 Immediately before loading samples onto the sequencer combine the following Per each sample take 9 ul of formamide 0 18 ul CE standards provided in the kit Prepare Master Mix for all the samples to be analyzed plus one extra In 96 well plate for CE analysis combine 9 ul of Master mix with 1 ul of purified labeled FACTORIAL cDNA Step IX The ABI capillary electrophoresis machine will produce a fsa file that is ready for analysis u
28. nsion Product 24 IX Proteinase ope aieo acad nea ache 24 X Capillary Electrophoresis ead ee ac ca RE 25 Frequently Asked Questions 27 Troubleshooting Guide 28 Trademarks and Patent 30 End user license agreement 30 Product G ararnlt 6 5c ox he n Ee e rt et sd et ie Is 30 Appendix A Settings Used on the ABI 3130 XL Genetic Analyzer 31 Appendix B Promoter Elements and References 32 Introduction Transcription factors TFs comprise classes of proteins that bind genomic regulatory elements and modulate gene transcription Signals that control gene expression are often executed through coordinated changes in the activities of transcription factors Analyzing the functional status of transcription factors is crucial to identifying signal transduction pathways that determine cell behavior Of the few tools currently available to researchers who seek to study cell regulation at the level of transcription factor activities none is geared towards quantitative high content assessments Assays that make use of reporter gene constructs e g luciferase reporters can be used to analyze only one or two transcription factors at a time In contrast DNA binding assays can potentially be used to assess multiple transcription factors but pro
29. o minimize RNase contamination The amount of RNA used for each sample should fall within the suggested range indicated in the reverse transcription protocol 0 5 to 5 ug It is not necessary to adjust your samples to contain equal amounts of RNA for each RT reaction The results will be internally normalized during the data analysis procedure by reporting relative levels instead of absolute levels of expression If your cells have not been efficiently transfected with the FACTORIAL Reagent you should use more RNA for the DNase treatment than you would if your cells had a high transfection efficiency This protocol has been optimized for DNase provided with the FACTORIAL kit but other DNases have been tested and can be used with little or no modification of this protocol follow manufacturer s instructions in this case Protocol Note Amounts in the following protocol are given per single reaction We advise preparing a Master Mix for all the samples plus one extra reaction 1 4 Assemble the following reaction 0 5 to 5 ug of each RNA sample from Section III Adjust the volume to 17 ul using RNase free water 2 ul 10X DNase buffer 2 ul DNase Final reaction volume 20 ul Incubate for 30 minutes at 37 C Inactivate DNase by adding 2 ul of 50 mM EDTA and heating the tubes at 75 C for 15 minu tes Note If EDTA is not added after the DNase treatment the RNA may be chemically degraded in future steps
30. reatment step removes the impurities in the reaction that might clog the capillary electrophoresis machine Purification also removes salts that can interfere with the correct operation of the capillary electrophoresis machine Note The primer extension product has been labeled with a light sensitive fluorophore so take care to shield the DNA from light Protocol 1 Dilute the provided Proteinase K solution 1 30 in water 2 Add 1 ul of the diluted solution to the 26 ul processed primer extension reaction from Section VIII 3 Incubate at 37 C for 1 hour in the dark 4 Use Performa DTR gel filtration cartridges Edge Biosystems cat 42453 or PCR Purification columns Qiagen cat 28104 to remove proteins and salts You may store the purified labeled PCR product at 20 C in the dark until you are ready to proceed 24 X Capillary Electrophoresis Capillary electrophoresis separates and detects the amount of transcripts generated by the FACTORIAL Transcription Factor Reporter constructs Notes Capillary electrophoresis CE utilizes an ABI sequencer 3130xl or similar and is usually performed in a sequencing facility Please be advised that the data from other types of CE analyzers cannot be analyzed using our software If you are using a sequencing facility to analyze your DNA fragments please consult them regarding the best way to have your samples processed We recommend bringing this booklet to them and calli
31. riance in transfection efficiency and transcription profile Different transfection reagents produce different results with various cell lines A set of reagents and methods should be used that have been proven to be effective for transfecting plasmid DNA into the desired tissue culture cell line These transfection reagents and methods should be optimized to achieve the highest percentage of transfected cells while maintaining good cell viability We recommend that you follow the manufacturer s suggestions for optimization of your transfection To determine the percentage of cells transfected with a specific transfection protocol we suggest using a reporter plasmid containing B gal eGFP or another reporter to assess the percentage of transfection After transfection cell viability should be assessed via a method of your choice Depending on transfection efficiencies between 2 x 10 and 3 x 10 cells are plated for transfection Please consult Table 2 for general guidelines regarding the transfection protocol As a general rule 10 000 transfected cells are needed for each replicate in the analysis 15 Table 5 Recommended number of cells to plate for transfection of cells for transfection Plate format Amount of FACTORIAL transfected cells protocol for transfection reagent used 5 300 000 6 well dish 1 ug 10 200 000 6 well dish 1 ug 20 150 000 6 well dish 1 ug 30 100 000 12 well dish 0
32. s Mol Biol 2005 Feb 93 2 5 99 105 Review PMID 15860251 PubMed indexed for MEDLINE amp Ikeda Kawaguchi Kamekura S Ogata N Mori Y Nakamura S Chung SOXRE SOX family SOX 2 4 9 9 UI Distinct roles of Sox5 Sox6 and Sox9 in different stages of chondrogenic differentiation gene transcription differentiation J Bone Miner Metab 2005 23 5 337 40 Review No abstract available PMID 16133682 PubMed indexed for MEDLINE SP1RE SP1 SP1 Zhao C Meng Sp1 like transcription factors are regulators of embryonic embryonic development development in vertebrates Dev Growth Differ 2005 May 47 4 201 11 Review PMID 15921495 PubMed indexed for MEDLINE SREBPRE SREBP SREBP a c lipid homeostasis Eberle D Hegarty B Bossard P Ferre P Foufelle F SREBP transcription factors family master regulators of lipid homeostasis Biochimie 2004 Nov 86 1 1 839 48 Review PMID 15589694 PubMed indexed for MEDLINE cellular adhesi n Mulholland DJ Dedhar S Coetzee GA Nelson CC Interaction of nuclear receptors TCFRE TCF4 LEF1 LEF1 issus merpHe enesis with the Wnt beta catenin Tcf signaling axis Wnt you like to know Endocr Rev 2005 B catenin Dec 26 7 898 915 Epub 2005 Aug 26 Review PMID 16126938 PubMed indexed for MEDLINE f SMAD 3 Lee KY Bae SC TGF beta dependent cell growth arrest and apoptosis J Biochem Mol TORERE TGFB
33. s and cell cycle control Gene E Area differentiation 2000 Dec 30 260 1 2 1 12 Review PMID 11137286 PubMed indexed for MEDLINE BRE SMAD family BMP4 differentiation of osteoblasts Zhang J Li L BMP signaling and stem cell regulation Dev Biol 2005 Aug 1 284 1 1 11 Review PMID 15963490 PubMed indexed for MEDLINE Conkright MD Montminy CREB the unindicted cancer co conspirator Trends Cell CRE CREB ATE CREB1 forsk lin survival proliferation and glucose Biol 2005 Sep 15 9 457 9 Review PMID 16084096 PubMed indexed for Family cAMP metabolism MEDLINE C EBP p Buck Chojkier Signal transduction in the liver C EBPbeta modulates cell DEBERE family Omne resporises proliferation and survival Hepatology 2003 37 4 731 8 Review No abstract available PMID 12668962 PubMed indexed for MEDLINE RAR y me Sharoni Y Danilenko M Dubi N Ben Dor A Levy J Carotenoids and transcription Arch RARE giirerentiation Biochem Biophys 2004 Oct 1 430 1 89 96 Review PMID 15325915 PubMed Retinoic Acid indexed for MEDLINE ERE ER ER a ESR1 growth differentiation Geserick C Meyer HA Haendler B The role of DNA response elements as allosteric Estradiol and homeostasis maintenance modulators of steroid receptor function Mol Cell Endocrinol 2005 May 31 236 1 2 1 7 Review PMID 15876478 PubMed indexed for MEDLINE ET
34. sing the ATTAGRAPH Software Optional service note If you do not have an access to Genetic Analyzer and would like to use our services you can submit your samples for CE analysis and ship them to us according to the following instructions EL Sample preparation and shipping instructions Your samples should be fully processed and purified by recommended gel filtration or PCR purification columns For PCR purification columns from other manufacturers we suggest that you check column specifications and make sure that your smallest DNA fragment 109 b will be fully recovered after purification Please submit at least 5 ul aliquots of your samples for CE analysis Place aliquots in polypropylene PCR strips preferably with attached flat caps or you may choose to use PCR plates of suitable format 24 48 or 96 well Close tubes tightly or seal the plate with aluminum foil cover Label tubes with numbers on top and the side Each of your samples will be assigned the number with which you label it For samples on PCR plates each sample will be labeled according to well position A1 for example Wrap the tubes or plate in foil to shield from light Fill out the CE request form download from our web site and email it to us at info attagene com also include the hard copy with the package Ship samples on blue ice by overnight delivery to Attagene Inc 7030 Kit Creek Rd Morrisville NC 27560 Please refer to ATTAGRAPH Software
35. sity of the 100 ng DNA Mass Ladder band that have been highlighted by the red arrow above If there is less than 100 ng of PCR product the PCR should be repeated and a new gel should be run If the problem of not enough DNA persists see the Troubleshooting guide for further action The PCR products can be stored at 20 C until you are ready to proceed 99 Vil Labeling FACTORIAL DNA by Primer Extension After sufficient FACTORIAL specific PCR product is available it may be labeled via a primer extension reaction This labeling allows detection and quantification of the FACTORIAL reporter cDNA by using the capillary electrophoresis conditions described in Section X Notes Use aerosol tips and gloves during the assembly of the primer extension reaction The conjugated fluorophore is sensitive to light so attempt to minimize the time that the oligonucleotide and primer extension products are exposed to light We suggest using a thermocycler for the labeling reaction Protocol Important If the amount of the PCR product from the previous step is between 100 and 250 ng use 1 10 of that for labeling if the amount exceeds 250 ng use 1 20 of that PCR product for labeling Note Amounts in the following protocol are given per single reaction We advise preparing a Master Mix for all the samples plus one extra reaction 1 Assemble labeling reaction 19 25 ul water 2 5 ul 10X PCR buffer 0 5 ul dNTPs 10mM of eac
36. table for a reverse transcription reaction It will be necessary to isolate at least 0 5 ug of total RNA from the transfected cells in a final volume of no more than 80 ul The total amount of RNA needed will depend on the transfection efficiency of the cells the lower the transfection efficiency of the cells the greater the amount of RNA will be needed to obtain a reproducible FACTORIAL analysis Once isolated the RNA should be analyzed using a spectrophotometer to assess the purity and amount of RNA recovered By taking absorbance readings at 260 nm and 280 nm the purity of the RNA can be evaluated The 260 280 ratio should be 2 0 A ratio less than 1 75 or greater than 2 0 suggests that there may be contaminants present The RNA from this step may be stored at 80 C until you are ready to proceed IV DNase Treatment If your RNA isolation protocol included an on column DNase treatment step you can skip this step and proceed directly to Section V RNA isolated from the transfected and treated cells must be digested with DNase to remove any residual FACTORIAL Reagent that may have contaminated the sample It is necessary to remove as much of the transfected FACTORIAL reagent as possible from the RNA sample to ensure that the plasmid DNA does not interfere with the subsequent analysis 17 Notes Be sure to use RNase free tips tubes and solutions for this section of the protocol Gloves should be worn at all times t
37. ted usage license for research use only FACTORIAL30 products are not intended for any treatment or diagnostic purposes in human subjects It is the user s responsibility to comply with the applicable laws and government regulations should these products be used for other purposes than research FACTORIAL30 products can not be used for commercial purposes or resale reproduction or modifications of any kind as a whole or any parts of them By purchasing our kits user understands that Attagene Inc can only guarantee their performance if used as described in this Manual Attagene Inc does not assume responsibility for any damage or loss directly or indirectly associated with purchasing of our products We recommend that users of the FACTORIAL product line will exercise all due care while handling our products NIH or other guidelines that applicable to recombinant DNA experiments should be closely followed Product Guarantee Attagene Inc guarantees the performance of these products if used as described in this Manual If any of our products fail to perform satisfactorily due to reasons other than misuse Attagene will replace the product free of charge issue the credit or fully reimburse your account Please consult our Technical support in case if you have questions or concerns about our products 30 APPENDIX A Settings used on the ABI 3130 XL Genetic Analyzer for fragment analysis of the FACTORIAL Reagent Mame Val
38. tire manual before beginning your experiment Materials and Equipment Required FACTORIAL30 Kit Components Cat numbers FPS 03020A 03020B 03020C 03020D 03020E 03020F 03020Explorer FACTORIAL30 XL Kit Components Cat numbers FPS 03020A XL 03020B XL 03020C XL 03020D XL 03020E XL 03020F XL 03020 XL Explorer Reagents provided in each kit are sufficient to perform 20 FACTORIAL assays which corresponds to 20 transfections using 6 well plate format Table 4 Reagents provided in the FACTORIAL30 kits FACTORIAL30 FACTORIAL30 FACTORIAL30 XL Reagent name Explorer 1 FACTORIAL Reporter Library 1 ug ul 25 ul 10 ul each 25 ul 2 Primer for reverse transcription RT primer 0 5 ug ul 45 ul 45 ul 45 ul 3 FACTORIAL specific PCR Primer MIX 20 pM ul each 90 yl 90 90 ul 4 FACTORIAL specific Labeling Primer 20 pM ul 25 ul 25 ul 25 ul 5 Hpal restriction enzyme 25 ul 25 ul 25 ul 6 Proteinase K solution 5 ul 5 ul 5 ul 7 FACTORIAL Calibration Standards 12 ul 12 ul 12 ul 8 Capillary Electrophoresis Standards 18 ul 18 ul 18 ul 9 RNAse free DNase 90 ul 10 10X DNase buffer 180 ul 11 50 mM EDTA 90 ul 12 dNTP Mix 10mM of each dATP dCTP dGTP and dTTP 90 ul 13 50 mM MgCl 20 ul 14 10X First strand RT buffer 90 ul 15 M MLV reverse transcriptase 200 units ul 45 ul 16 10X PCR
39. ue Range 0 18 650696 Poly_Fill_ ol 8500 6500 35000 steps Current Stability 50 0 2000 uAmps z PreRun voltage 150 0 15 Pre Run Time 190 1 1000 sec Injectian_Voltage 12 1 15 k olis x Injection Time 23 1 500 SEF Voltage Number Of Steps 20 1 100 nk Voltage Step Interval 15 1 60 sec 2 Data Delay Time 80 1 3500 sec Run Voltage 0 0 0 15 Kvols Run_Time 2000 300 14000 sec 31 APPENDIX Promoter Elements and References T ipti M Factor Transcription Factors Biological Function Reviews Family Name Inducers Tested xenobiotic response neurogenesis Swanson HI DNA binding and protein interactions of the AHR ARNT heterodimer that AhRRE AhR AhR dioxin hypoxia cardiovascular development facilitate gene activation Chem Biol Interact 2002 Sep 20 141 1 2 63 76 Review angiogenesis hypoxia PMID 12213385 PubMed indexed for MEDLINE JNICBatbiwavsoroliteratioh Wagner EF Matsuo K Signalling in osteoclasts and the role of Fos AP1 proteins Ann AP1 c jun c fos c jun c fos PMA A Rheum Dis 2003 Nov 62 Suppl 2 183 5 Review No abstract available PMID 14532157 PubMed indexed for MEDLINE Hilger Eversheim K Moser M Schorle H Buettner R Regulatory roles of AP 2 embryonic morphogenesis transcription factors in vertebrate development apoptosi
40. ure format FACTORIAL PCR samples can be stored frozen at 20 C adde Vil Labeling This step allows detection of the FACTORIAL Reporter cDNAs during capillary electrophoresis Labeling is performed via a primer extension reaction using FACTORIAL specific primers coupled with a fluorescent dye such as 6 FAM Vill Processing This step allows separation of the individual FACTORIAL Reporter cDNAs during capillary electrophoresis Processing is achieved by digestion of the labeled cDNA with Hpa restriction endonuclease IX Purification This step provides optimal conditions for capillary electrophoresis that is sensitive to certain impurities present in the samples such as salt and proteins Sufficient purification is achieved by treatment of the samples with proteinase K following desalting using gel filtration spin columns X Capillary electrophoresis This step is required to detect and quantify individual FACTORIAL Reporter cDNAs Electrophoresis is usually performed in a DNA sequencing facility using standard ABI capillary electrophoresis instrument i e ABI 3100 or ABI 3130 Please consult your Institution s Sequencing Facility to determine if they perform fragment analysis service Alternatively labeled samples can be submitted for analysis to one of the designated sequencing facilities see the corresponding section of this manual On the electrophoregramm the individual FACTORIAL Reporter cDNAs are identifia
41. vide limited biological information because DNA binding is only one of many determining factors in the regulation of transcription factor activity Attagene has conceptualized and developed a simple homogeneous assay termed FACTORIAL that enables assessment of the activities of multiple transcription factors in a single experiment Our own studies using this new technology have demonstrated consistent reproducibility and ease of application in different biological settings The FACTORIAL assay is now being offered to a broad academic research community please refer to end user licensing agreement on page 30 of this Manual With the FACTORIAL30 assay you can analyze basal and induced transcription factor activity profiles in a variety of commonly used cell lines assess the function of a gene of interest in the context of its effects on activities of multiple transcription factors investigate mechanisms of action of biologically active agents including chemical compounds peptides siRNAs and dominant negative variants of proteins explore signal transduction pathways underlying alterations in gene expression profiles generated by microarray hybridization 1 Romanov S Medvedev A Gambarian M Poltoratskaya N Moeser M Medvedeva L Gambarian M Diatchenko L Makarov S Homogenous reporter system enables quantitative functional assessment of Multiple transcription factors Nat Methods 2008 Mar 5 3 253 60 Epu
Download Pdf Manuals
Related Search