Oncogene Profiler PAC
Contents
1. peu wo NOH paa INSA Tala 9XOG WXIH ZINM O INL 145 5 6 142644 Izd VIIN PPW ISW MVA IOND NAI gda IS3HO INI Lum H9Lns ous 91d srw erm rr gaia 9 ood PPO Noga IPV ANL HSl4ns qus QIVA rwd 914 ofu p IN3W e voni sunf 2480 TID Ald 1484 IHdO pav THA L4SPWi Heldns 1seo vio Zuna zord quw unto m 29 riya z qv SAVA NIL SIS NON ZWdW sivont 1 9Z u 5 save HOYA S IHO ZagZI ZAVA e awu giis Vous uero run livont gum 10 W94 PIMA 1 104 IO bnro aos 94101 LAV maodd quq gaa sama ayo pono dDdd AON IXW xmu cno paoa 81 91 51 iat SI ZI II 01 6 8 2 9 g 2 1 6 e3iy u 5o5uo 2 Z A m OO s Genemed Biotechnologies Inc 458 Carlton Court South San Francisco 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com2. Genbank Gene name LIBC B10 AF143679 Homo sapiens lost in inflammatory breast cancer tumor suppressor protein LIBC mRNA LUCA 1 C10 U03056 Homo sapiens putative tumor suppressor LUCA1 HYALT LUCA15 D10 U23946 Human putative tumor suppressor LUCA15 mRNA LUCA 2 E10 NM 003773 Homo sapiens hyaluronoglucosaminidase 2 HYAL2 mRNA LUCA 3 F10 AF040710 Homo sapiens hyaluronidase LUCA 3 mRNA 151 10 77753 H sapiens TROP 2 gene M4S1 H10 M32325 Human adenocarcinoma associated antigen KSA mRNA mad 10 106895 Human antagonizer of myc transcriptional activity Maf c maf J10 NM_005360 Human v maf musculoaponeurotic fibrosarcoma avian oncogene MAFB K10 AF134157 Homo sapiens MAFB Kreisler basic region leucine zipper transcription factor MAFB mRNA MAFG L10 U84249 Homo sapiens basic leucine zipper transcription factor MafG MAPKAPK3 M10 NM 004635 Homo sapiens mitogen activated protein kinase activated protein kinase 3 MAPKAPK3 Mrna Mas c mas N10 M13150 Human mas proto oncogene gene MASL1 O10 NM_004225 Homo sapiens MFH amplified sequences with leucine rich tandem repeats 1 MASL1 mRNA max A X60287 595058 Human max gene MCC B11 M62397 Human colorectal mutant cancer protein mRNA MDA 7 NM 006850 Homo sapiens suppression of tumorigenicity 16 melanoma differentiation MDA 7 mRNA MDM2 D11 Z12020 Human p53 associated gene MEL 11 553268 MEL RAS related human Genomic mRNA 1980 nt MEN1 11 1093236 Human meni
3. class receptor tyrosine kinase FMS c fms L07 X03663 Human c fms proto oncogene fos c fos M07 00650 M16287 Human Proto oncogene FOSL1 N07 X16707 Human fra 1 mRNA FOSL2 O07 X16706 Human fra 2 mRNA 1 P07 U58975 Homo sapiens proto oncogene 1 mRNA FRAT2 08 AF062739 Homo sapiens GSK 3 binding protein FRAT2 FRAT2 Fus 2 B08 AF040706 Homo sapiens putative tumor suppressor protein Fus 2 Gene 21 C08 AF040708 Homo sapiens candidate tumor suppressor gene 21 protein Gene 26 D08 AF040709 Homo sapiens putative tumor suppressor gene 26 protein alpha 2 delta calcium channel subunit Mrna gli E08 NM 005269 Homo sapiens glioma associated oncogene homolog zinc finger protein GLI mRNA gli2 F08 NM 005270 Homo sapiens GLI Kruppel family member GLI2 GLI2 GLI3 G08 M57609 M34366 Human DNA binding protein GLI3 mRNA GRO1 H08 X12510 Human mRNA for melanoma growth stimulatory activity MGSA HCK 108 M16591 Human hemopoietic cell protein tyrosine kinase HCK gene hdlg 1 J08 U13897 Human homolog of Drosophila discs large protein isoform 1 HEMK K08 AF131220 Homo sapiens HEMK homolog HEMK mRNA HLF L08 M95585 Human hepatic leukemia factor HLF mRNA Hox11 M08 NM 005521 Homo sapiens homeo box 11 T cell lymphoma 3 associated breakpoint HOX11 mRNA HPV E6 N08 K02718 Human papillomavirus type 16 HPV16 complete genome HPV E7 O08 K02718 Human papillomavirus type 16 HPV16 complete genome hsReg P08 U94585 Homo sa
4. mRNA EXIL2 AF000416 Homo sapiens EXT like protein 2 EXTL2 mRNA EXIL3 L06 AF083551 Homo sapiens exostosin EXTL3 mRNA FAT M06 X87241 H sapiens mRNA for hFat protein FER N06 J03358 Human tyrosine kinase FER mRNA FES c fes O06 X52192 Human c fes fps proto oncogene FGF 3 P06 NM 005247 Human fibroblast growth factor 3 murine mammary tumor virus integration site v int 2 oncogene homolog FGF3 Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Coord Genbank Gene name FGF 4 A07 NM_002008 Human fibroblast growth factor 4 heparin secretory transforming protein 1 Kaposi sarcoma oncogene FGF4 FGF 6 B07 X63454 H sapiens hst 2 FGF 6 mRNA FGFR C07 M34185 Human fibroblast growth factor receptor FGFr transmem brane form mRNA complete cds FGR D07 M19722 J03429 Human Proto oncogene FHIT E07 NM_002012 Human fragile histidine triad gene FHIT FKHL1 F07 X74142 H sapiens HBF 1 mRNA for transcription factor FLI 1 G07 X67001 544250 H sapiens HUMFLI 1 mRNA FLT 1 H07 AF063657 Homo sapiens vascular endothelial growth factor receptor Flt 1 107 AF063657 Homo sapiens vascular endothelial growth factor receptor FLT1 mRNA 107 726652 564785 FLT3 mRNA for FLT3 receptor tyrosine kinase Fit4 07 68203 H sapiens mRNA for FLTA
5. salmon e 100u 5X RT Buffer purple e 100ul dH 0 natural Profiler Array Chemiluminescence Detection Kit Store at 4 15ml CDP Svzar Substrate 30ml 5X Blocking Buffer 50ml 10 Antibody Wash Buffer I 50ml 10 Antibody Wash Buffer II 30ml 10X Detection Buffer e 15 Alk Phosphatase Conjugate orange Additional Materials Provided 1 Profiler Grid 1 Product User Manual Tsee Product Analysis Certificate provided with each kit for complete list of genes spotted on the array Genemed Biotechnologies Inc Page 5 Profiler Array Nonradioactive System ADDITIONAL MATERIALS amp EQUIPMENT Additional Materials Required Sheared salmon testes DNA Sigma Cat D7656 Hyperfilm ECL Film Amersham Cat RPN31144 Plastic wrap e g Saran Wrap or plastic sheet protectors Commercial pouch sealer or equivalent Additional Materials Recommended Trizol Life Technologies Cat 15596 018 or equivalent Additional Equipment IV Thermal cycler or water bath Temperature controlled Shaking or Rotating platform Optional Hybridization oven and bottles Stratagene Cat 401031 Optional Alpha Innotech FluorChem or equivalent INSTRUCTIONS FOR USE Isolation of total RNA from cells or tissues Total RNA be isolated from tissues or cells using standard methods e g Trizol or equivalent You can use poly A RNA if you wish however we have found that equivalent results can be ac
6. B05 M18288 Adenovirus type 40 E1A and E1B genes E2F 1 C05 M96577 Human E2F 1 pRB binding protein gene eat3 D05 X12795 Human v erbA related ear 5 gene EGFR E05 NM 005228 Human epidermal growth factor receptor Egr 1 F05 NM 001964 Human early growth response 1 EGR1 gene EIF3S6 G05 U62962 Human Int 6 mRNA EIF4B H05 NM 001417 Human eukaryotic translation initiation factor 4B EIF4B ELE1 105 X71413 H sapiens ELE1 mRNA ELK1 J05 M25269 Homo sapiens tyrosine kinase ELK1 oncogene mRNA ELK3 K05 736715 H sapiens mRNA for Net transcription factor EMP1 L05 U77085 Human epithelial membrane protein CL 20 mRNA EMS1 M05 M98343 Homo sapiens amplaxin EMS1 mRNA EPHA1 N05 M18391 Human tyrosine kinase receptor eph mRNA EPHA3 O05 M83941 Human receptor tyrosine kinase HEK mRNA ERBAL2 P05 X12794 Human v erbA related ear 2 gene ERbB2 c erbB2 06 X03363 Human c etb B 2 mRNA ERBB3 B06 M34309 Human epidermal growth factor receptor HER3 mRNA ERBB4 C06 L07868 Homo sapiens receptor tyrosine kinase ERBB4 gene Ets1 D06 J04101 Human erythroblastosis virus oncogene homolog 1 ets 1 Ets2 E06 NM 005239 Homo sapiens v ets avian erythroblastosis virus E26 oncogene homolog 2 ETS2 mRNA EIV3 F06 L16464 Human ETS oncogene PEP1 mRNA ETV6 G06 U11732 Human ets like gene tel mRNA EVI H06 582592 Evi 1 Evi 1 protein 3 region deletion region EWSR1 106 X66899 H sapiens EWS mRNA EXT2 106 NM 000401 Homo sapiens exostoses multiple 2 2
7. IMPLIED ARE PROVIDED INCLUDING WARRANTIES FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE UNDER NO CIRCUMSTANCES SHALL GENEMED BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OF THE MATERIALS 2000 Genemed Biotechnologies Inc Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com gt 6 Genemed Biotechnologies Inc Profiler Array Nonradioactive System User Manual Version 2 2 STORAGE Store Box 1 at room temperature RT Box 2 at 20 C and Box 3 at 4 C Please note that the Substrate is light sensitive and should be stored in the dark FOR RESEARCH USE ONLY PRT0088 Profiler Array Nonradioactive System TABLE OF CONTENTS L Introduction 3 IL Package Contents 5 IIl Additional Materials amp Equipment 6 IV Instructions for Use 6 V Troubleshooting Guide 10 Appendix 11 TRADEMARKS Profiler is a trademark of Genemed Biotechnologies Inc CDP ar is a registered trademark of Tropix Inc GenBank is a registered trademark of The United States Department of Health and Human Services FluorChem is a trademark of Alpha Innotech Corporation Hyperfilm is a trademark of Amersham Pharmacia Biotech Saran Wrap is a registered trademark of S C Johnson WARRANTY GENEMED BIOTECHNOLOGIES INC WARRANTS THAT THE MATERIALS SOLD MEET GENEME
8. RAN member RAS oncogene familyHomo sapiens RAN member RAS oncogene family RAN mRNA RARRES3 H15 AF060228 Homo sapiens retinoic acid receptor responder 3 RARRES3 Ras Ha ras 115 M30708 Synthetic human c Ha ras gene encoding p21 protein Ras Ki ras 115 L00044 00653 Human cellular c Ki ras2 proto oncogene Ras N ras K15 M14307 Synthetic N ras gene Rb p110 L15 M28419 Human retinoblastoma susceptibility protein mRNA Rb2 p130 M15 X74594 Human Rb2 p130 protein RBX1 N15 AF140598 Homo sapiens ring box protein 1 RBX1 mRNA RDA32 O15 AF061836 Homo sapiens putative tumor suppressor protein RDA32 RECK P15 E13833 Human tumor suppressor gene RECK gene Rel c rel A16 002908 Homo sapiens v rel avian reticuloendotheliosis viral oncogene homolog REL mRNA Ret B16 X12949 M57464 Human ret proto oncogene for tyrosine kinase RhoA C16 L25080 Human GTP binding protein rhoA RON D16 X70040 H sapiens RON mRNA for tyrosine kinase Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Coord Genbank Gene name Ros 1 E16 NM_002944 Homo sapiens v ros avian UR2 sarcoma virus oncogene homolog 1 ROS1 mRNA shb F16 X75342 H sapiens SHB mRNA she G16 U73377 Human p66shc SHC mRNA Ski c ski H16 X15218 Human ski oncogene gene Smad4 16 U44378 Human homozygous del
9. www genemed com Oncogene Profiler Gene List CONTROLS beta actin Ubiquitin Coordinates A1 amp 19 P1 10 19 amp 24 M24 N24 O24 amp 19 P2 amp 11 The following gene list is ordered alphabetically for your convenience Genbank X00351 J00074 M10278 M26880 Gene Coord Genbank Gene name 101F6 Col AF040704 Homo sapiens putative tumor suppressor protein 101F6 123F2 D01 AF040703 Homo sapiens putative tumor suppressor protein 123F2 Abl 2 E01 NM_005158 Human v abl Abelson murine leukemia viral oncogene homolog 2 arg Abelson related gene ABL2 gene AF 4 F01 X83606 H sapiens AF 4 gene G01 NM_005163 Human v akt murine thymoma viral oncogene homolog 1 Akt2 H01 NM_001626 Homo sapiens v akt murine thymoma viral oncogene homolog 2 Akt3 101 AF135794 Human AKT3 protein kinase alpha catenin J01 S78736 CTNNA1 tumor suppressor gene alpha catenin aml 1 K01 L34598 Homo sapiens clone PEBP2aB aml1 acute myeloid leukemia 1 oncogene AML1 APC L01 M74088 Human APC gene ARHA M01 L09159 Homo sapiens RHOA proto oncogene multi drug resistance protein mRNA 3 end AXL N01 M76125 _061699 Human tyrosine kinase receptor axl BAM22 O01 L13939 Homo sapiens beta adaptin BAM22 BARD1 A02 NM_000465 Homo sapiens BRCA1 associated RING domain 1 BARD1 BCD ORF1 B02 U51869 Human proto oncogene Bcd orf1 and orf2 BCD ORF2 C02 U51869 Human proto oncogene Bcd orf1 and orf2 Bcl2 D02 M14745 Human b
10. 000 Problem hybridization signals weak or absent Is the problem with your RNA quality Be sure to check the quality of your RNA Section IV A eAre the kit reagents working properly Strip the membrane and use the control K562 RNA provided to confirm that reagents and materials are working propetly e Did you hybridize the membrane at the correct temperature Check to make sure the hybridization temperature is set at 52 C e How long was the hybridization time An overnight hybridization 12 hours is necessary for optimal binding of the probe to the array How much CDP Star substrate did you use Make sure that the substrate is evenly distributed over the membrane during detection Step E5 e How long was the exposure Try several exposures for varying lengths of time This is necessary for ob taining optimal results Page 10 Genemed Biotechnologies Inc INOL LOS 53005 unoe eyeg Profiler Array Nonradioactive System APPENDIX Array Orientation VI Page11 Genemed Biotechnologies Inc VI APPENDIX continued Solution Dilution Recipes enough for two array membranes 250 ml of 2X SSC 0 5 SDS To 212 5 ml of deionized H2O add 25 ml of 20X SSC stock solution and 12 5 ml of 10 SDS stock solution Shake well before use 250 ml of 0 1X SSC 0 5 SDS To 236 25 ml of deionized H2O add 1 25 ml of 20X SSC stock solution and 12 5 ml of 10 SDS stock solution Shake well before use 75 ml of 1X Blo
11. D S PERFORMANCE SPECIFICATIONS FROM THE TIME OF SHIPMENT UNTIL THE EXPIRATION DATE IF STORED UNDER THE RECOMMENDED CONDITIONS NO OTHER WARRANTIES OR GUARANTEES EXPRESSED OR IMPLIED ARE PROVIDED IN CLUDING WARRANTIES FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PUR POSE UNDER NO CIRCUMSTANCES SHALL GENEMED BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OF THE MATERIALS 2000 Genemed Biotechnologies Inc Page2 Genemed Biotechnologies Inc Profiler Array Nonradioactive System I INTRODUCTION With the advent of macroarray technology studying differential gene expres sion has become relatively easy and routine The expression profiles of hun dreds of genes can be studied simultaneously in a single array experiment Typically array experiments require the use of radioactive cDNA probes However working with radioactively labeled probes is a hassle requiring time consuming procedures Furthermore radioactively labeled cDNA arrays tend to generate high levels of background The Profiler Array provides researchers with an alternative approach a hassle free nonradioactive array system that employs digoxigenin DIG 11 dUTP labeled cDNA and chemiluminescent detection With this method you can obtain semiquantitative results in just 1 2 days compared to 4 7 days that radioactive based methods demand Our first array the Star Profiler Array contains 373 genes an exclusive set of the most popular and well s
12. F D WAG RRA 4 Geneme Biotechnologies Inc PRODUCT ANALYSIS CERTIFICATE Oncogene Profiler Array Nonradioactive System FOR RESEARCH USE ONLY CATALOG 05 101 Description The Oncogene Profiler Array Nonradioactive System employs digoxigenin DIG 11 dUTP labeled cDNA and chemiluminescent detection Each Oncogene Profiler Array contains 295 human genes which have been spotted onto a positively charged nylon membrane The spots on the array consists of popular and well studied genes that have been implicated in cancer and tumorigenesis Enough reagents are provided for four hybridizations Package Contents Profiler Array and Hybridization Kit 2 Oncogene Profiler Arrays 25 ml Hybridization Buffer 60 ml 20X SSC 60 ml 10 SDS Profiler Array cDNA Synthesis Kit 10 ug Control K562 Total RNA 15 Random Primer Mix 15 dNTP Mix 100 pl 20 Profiler Array Chemiluminescence Detection Kit 15 ml CDP Svzar Substrate 30 ml 5 Blocking Buffer 50ml 10X Antibody Wash Buffer I 50ml 10X Antibody Wash Buffer II 30ml 10X Detection Buffer Additional Materials 1 Profiler Grid 1 Product User Manual Storage Store the Profiler Array and Hybridization Kit at room temperature Profiler Array cDNA synthesis Kit at 20 C and Profiler Array Chemiluminescence Kit at 4 C Shelf Life 1 year from date of receipt under proper storage conditions Shipping Conditions Blue ice 4 C or room temperature Quality Control A sample of t
13. Fax 650 952 1060 Email info genemed com www genemed com Coord Genbank Gene name VAV3 D19 NM 006113 Homo sapiens vav 3 oncogene VAV3 mRNA VHL E19 NM 000551 Homo sapiens von Hippel Lindau syndrome VHL mRNA V SfC 19 D10652 Rous sarcoma virus genome wnt 1 G19 NM_005430 Human wingless type MMTV integration site family member 1 WNT1 gene WNT2 H19 X07876 Y00838 Human mRNA for irp protein int 1 related protein WNT5 119 L20861 Homo sapiens proto oncogene Wnt 5a mRNA Wnt7a J19 D83175 Homo sapiens WNT7a mRNA WT K19 X51630 Human Wilms tumor WT1 mRNA for zinc finger protein Krueppel like Yes c yes 119 15990 Human 1 Genemed Biotechnologies Inc 458 Carlton Court South San Francisco 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com NOTES Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com NOTES Trademark Profiler is a trademark of Genemed Biotechnologies Inc CDP Svar is a registered trademark of Tropix Inc WARRANTY GENEMED BIOTECHNOLOGIES INC WARRANTS THAT THE MATERIALS SOLD MEET GENEMED S PERFORMANCE SPECI FICATIONS FROM THE TIME OF SHIPMENT UNTIL THE EXPIRATION DATE IF STORED UNDER THE RECOMMENDED CON DITIONS NO OTHER WARRANTIES OR GUARANTEES EXPRESSED OR
14. Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Coord Genbank Gene name CRK c crk M03 NM 005206 Human v crk avian sarcoma virus CT10 oncogene homolog CRK gene CRK II N03 D10656 Human mRNA for CRK II CRKL O03 X59656 H sapiens crk like gene CRKL Cyclin D1 P03 M64349 Human cyclin D1 Cyclin D2 A04 001759 Human cyclin D2 CCND2 Cyclin D3 B04 M90814 Human D3 type cyclin CCND3 gene D10S170 C04 S72869 H4 D10S170 putative cytoskeletal protein DACH D04 AF102546 Homo sapiens dachshund DACH mRNA DAL1 E04 AF069072 Homo sapiens putative lung tumor suppressor DAL1 mRNA dbl F04 X12556 Human dbl proto oncogene DCC G04 X76132 Human DCC gene DDX1 H04 NM 004939 Homo sapiens DEAD H Asp Glu Ala Asp His box polypeptide 1 DDX1 DDX6 I04 D17532 Human mRNA for RCK dek J04 X64229 589712 H sapiens dek mRNA DLG2 K04 X82895 H sapiens mRNA for DLG2 Doc 1 L04 AF006484 Homo sapiens putative oral tumor suppressor protein doc 1 Doc 1R M04 NM_005851 Homo sapiens tumor suppressor deleted in oral cancer related 1 DPC4 SMAD4 N04 U44378 Human homozygous deletion target in pancreatic carcinoma DPH2L O04 S81752 DPH2L candidate tumor suppressor gene ovarian cancer critical region of deletion ElAc P04 M18288 Adenovirus type 40 E1A and E1B genes E1B19K A05 M18288 Adenovirus type 40 E1A and E1B genes E1B55K
15. are Labeled cDNA amp be pa DIG DIG id Hybridization Bag Hybridize overnight 712 hr E ig 2 US i SE p 2 3 Detect amp analyze Rr C am be 3 4 hr 5 min exposure to x ray film Total Time 1 2 days 24 28 hr Figure 1 provides a simplified overview of the Profiler procedure After labeling Profiler Arrays are hybridized overnight with DIG 11 dUTP labeled cDNA The next day the array membranes are washed and subsequently in cubated with Anti DIG Alkaline Phosphatase DIG AP Antibody Upon addition of the CDP Star chemiluminescent substrate provided detection of DIG AP can be obtained within minutes by exposing array membranes to x ray film or by using a chemiluminescent imaging system such as Alpha Innotech s FluorChem Imaging System Page 4 Genemed Biotechnologies Inc Profiler Array Nonradioactive System II PACKAGE CONTENTS See the front cover of this User Manual for appropriate storage conditions For Buffer Solutions amp Recipes see Appendix at the back of this User Manual Profiler Array Nonradioactive System Profiler Array and Hybridization Kit Store at room temp 2 Profiler Arrays 25ml Hybridization Buffer 60ml 20X SSC 60ml 10 SDS Array cDNA Synthesis Kit Store at 20 C 10ug Control K562 Total RNA yellow e 10 Reverse Transcriptase amber 15 ul Random Primer Mix blue 15 Probe Mix pink e 5 100mM DTT
16. cant 0 1X SSC 0 5 SDS washing solution and add 30 ml of 1X Blocking Buffer to each container 2 Incubate at room temperature for 30 min 3 Prepare anti DIG AP Antibody Mixture For each membrane mix 1 ml of 1X Blocking Buffer and 6 ul of anti DIG AP Antibody Mix well Add the 1 ml mixture to each container and incubate at room temperature for 30 min Note Be sure to spin the antibody conjugate for 5 minutes at maximum speed before diluting in 1X Blocking Buffer Some lots may contain aggregated conjugates that precipi tate and can cause a spotty background on the membrane 4 Wash the membranes at room temperature Remove the anti DIG AP Antibody Mixture Blocking Buffer and wash twice using 40 ml of 1X An tibody Wash Buffer I for 10 min per wash Then repeat washes using 40 ml of 1X Antibody Wash Buffer II for 10 min per wash 5 Dilute CDP Star Substrate in 1X Detection Buffer 1 30 for x ray film or 1 10 for Imager Incubate each membrane in 30 ml diluted substrate for 30 minutes at temperature 6 Using forceps remove each membrane from the diluted substrate Do not allow the membrane to dry We recommend placing the membrane on a plastic support such as a developed used x ray film Page 8 Genemed Biotechnologies Inc IV Profiler Array Nonradioactive System INSTRUCTIONS FOR USE continued 7 Immediately cover each membrane with plastic wrap e g Saran Wrap or plastic sheet protectors and remove exce
17. cking Buffer To 60 ml of deionized H2O add 15 ml of 5X Blocking Buffer stock solution Shake well before use 200 ml of 1X Antibody Wash Buffer I To 180 ml of deionized H2O add 20 ml of 10X Antibody Wash Buffer I stock solu tion Shake well before use 200 ml of 1X Antibody Wash Buffer II 180 ml of deionized H2O add 20 ml of 10X Antibody Wash Buffer II stock solution Shake well before use 100 ml of 1X Detection Buffer To 90 ml of deionized H2O add 10 ml of 10X Detection Buffer stock solution Shake well before use Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 650 952 0110 Toll Free USA amp Canada 1 877 GENEMED 436 3633 Fax 650 952 1060 Email infoOgenemed com www genemed com Technical Support Tel 650 952 0110 Toll Free USA amp Canada 1 877 GENEMED 436 3633 Fax 650 952 1060 Email tech genemed com
18. cl 2 BCL3 E02 M31732 Human B cell lymphoma 3 encoded protein bcl 3 Bcr F02 NM_004327 Human breakpoint cluster region BCR gene BECN1 G02 AF139131 Homo sapiens 1 BECN1 mRNA BIN1 H02 AF068916 Homo sapiens bridging integrator protein 1 isoform BIN1 13 BLYM 102 K01884 Human Blym 1 transforming gene Bmil J02 NM_005180 Homo sapiens murine leukemia viral bmi 1 oncogene homolog BMI1 BRCA1 K02 NM 000058 Human breast cancer 1 BRCA2 L02 U43746 Human breast cancer 2 Brush 1 M02 569790 Brush 1 tumor suppressor 3 region human breast epithelium mRNA Partial 1485 nt CIORF1 N02 001213 Homo sapiens chromosome 1 open reading frame 1 C1ORF1 c abl O02 NM 005157 Homo sapiens v abl Abelson murine leukemia viral oncogene homolog 1 1 CAN A03 X64228 589710 H sapiens 1 B03 NM 001753 Homo sapiens caveolin 1 CAV CBL c cbl C03 X57110 Human c cbl proto oncogene CBL B D03 U26710 Human cbl b CDC23 E03 AF053977 Homo sapiens cell division cycle protein 23 CDC23 CDH1 F03 Z13009 H sapiens mRNA for E cadherin Cdk4 G03 NM_000075 Human cyclin dependent kinase 4 CDK4 CHES1 H03 U68723 Human checkpoint suppressor 1 Ciaol 103 U63810 Homo sapiens WD40 protein Ciao 1 c mer 03 NM_006343 Homo sapiens c mer proto oncogene tyrosine kinase MERTK Cot D14497 Human mRNA fot proto oncogene protein CREBL2 1903 NM_001310 Homo sapiens cAMP responsive element binding protein like 2 Genemed Biotechnologies
19. ct to the average value of the housekeeping gene After normalizing each array compare spot intensities on separate arrays for each gene Generally a threefold increase or decrease in spot intensity is considered significant Genemed Biotechnologies Inc Page9 Profiler Array Nonradioactive System V TROUBLESHOOTING GUIDE The following list of tips is designed to help you troubleshoot problems that you may encounter when performing the procedure If you feel you need additional technical assistance please contact us by phone fax or email A Problem high background Was the membrane completely immersed in solution during the incubations Never let the membrane dry out and be sure membrane is completely im mersed during all incubations If desired paper clips can be used to clip mem branes to the bottom of the plastic containment during washing blocking detection steps Use extreme caution when using this method Was the membrane completely covered in the solutions during detection Be sure solutions completely cover the membrane during these steps Were the hybridization solutions completely dissolved Check Hybridization Buffer for visible precipitated particles s the film overexposed Try several exposures for varying lengths of time This is necessary for ob taining optimal results eHow much antibody did you add to the Blocking Buffer in Step Make sure the antibody is diluted 1 5
20. essor protein NOC2 NOEY2 P12 U96750 Homo sapiens putative tumor supressor NOEY2 mRNA NOV A13 X96584 H sapiens mRNA for NOV protein novH B13 78351 H sapiens novH gene exons 1 and 2 TPR C13 X66397 H sapiens tpr mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Coord Genbank Gene name ODC D13 M34158 J05271 Human ornithine decarboxylase ODC gene p107 E13 L14812 Human retinoblastoma related protein p107 gene 15 F13 L36844 Human clone 15 4 5 CDK inhibitory protein gene 16 13 127211 Human CDK4 inhibitor p16 INK4 gene p21 H13 L25610 Human cyclin dependent kinase inhibitor mRNA p53 113 K03199 Human p53 Tumor Antigen p57 J13 U48869 Human cdk inhibitor p57 KIP2 1 gene p73 K13 Y11416 H sapiens mRNA for P73 PAR 4 L13 NM_002583 Homo sapiens apoptosis response protein PAWR mRNA PAX2 M13 M89470 Human paired box protein PAX2 mRNA PBX1 N13 NM 002585 Homo sapiens pre B cell leukemia transcription factor 1 PBX2 O13 X59842 Human PBX2 mRNA PBX3 P13 X59841 Human PBX3 mRNA PDGF b c sis 14 02811 Human platelet derived growth factor chain PDGF B gene PDGFRL B14 NM 006207 Homo sapiens platelet derived growth factor receptor like PEP1 C14 L16464 Human ETS oncogene PEP1 mRNA 1 D14 AF144757 Hom
21. etion target in pancreatic carcinoma DPC4 gene SMAD5 J16 U73825 Homo sapiens Smad5 mRNA SMARCB1 K16 Y17118 Homo sapiens SNF5 INIT gene exon 1 and joined CDS SnoN L16 X15219 Human sno oncogene mRNA for snoN protein ski related SPI M16 X52056 Human mRNA for spi 1 proto oncogene SPINK1 N16 Y00705 Homo sapiens mRNA for pancreatic secretory inhibitor expressed in neoplastic tissue stc c fyn O16 NM 002037 Human FYN oncogene related to SRC FGR YES FYN src c Lyn P16 M16038 Human lyn mRNA encoding a tyrosine kinase Src c src A17 X59932 X71157 Human C SRC kinase SRP 1 B17 U20620 Homo sapiens SRP1 mRNA partial sequence ST13 C17 U17714 Homo sapiens putative tumor suppressor ST13 ST13 mRNA ST5 D17 545936 H TS1 HeLa tumor suppressor gene human revertant clone F2 mRNA Partial 2687 nt SUPT3H E17 AF064804 Homo sapiens transcription factor SUPT3H SUPT3H SUPT5H U56402 Human chromatin structural protein homolog SUPT5H SUPT6H G17 U46691 Human putative chromatin structure regulator SUPT6H TAL1 H17 M29038 Human stem cell protein SCL mRNA TCL 1 117 X82240 H sapiens mRNA for Tcell leukemia lymphoma 1 TCL 3 J17 M62626 Human T lymphocyte homeobox protein partial cds K17 U06935 Human thyrotroph embryonic factor TEF mRNA TEL L17 U11732 Human ets like gene tel mRNA TGFb RII M17 NM_003242 Homo sapiens transforming growth factor beta receptor 70 80kD TGFBR2 mRNA THRA N17 M24748 Human thyroid hor
22. hieved using total RNA Quantify your RNA samples by measuring the absorbance at 260 nm and 280 nm Total RNA samples that are free of proteins have an Ay ratio of 1 8 2 2 In addition you should check the quality of your RNA by agarose gel electrophoresis Run 0 5 2 0 ug of RNA on a denaturing formaldehyde agar ose ethidium bromide gel Two bright bands 285 and 185 rRNA should be observed at approximately 5 and 2 kb respectively These bands are sharply defined and visible at about a 2 1 ratio 285 185 Preparation of cDNA probe For best results we recommend using a thermal cycler for all incubation steps however if a thermal cycler is unavailable heating blocks may be used If de sired first perform prehybridization Step C 1 4 and then prepare RNA Step B 2 and cDNA synthesis mixture Step B 4 simultaneously 1 Preheat a thermal cycler or heating block or equivalent to 70 Page 6 Genemed Biotechnologies Inc IV Profiler Array Nonradioactive System INSTRUCTION FOR USE continued 2 For each total RNA sample combine the following components into a sterile 0 5 ml microcentrifuge tube 5 10 Ug Total 3 Random Primer Mix Bring each tube to a final volume of 10 Hl by adding dH O provided Mix well by pipetting and spin briefly in a microcentrifuge Keep on ice Heat samples at 72 C for 2 min Then reduce temperature to 42 C and incubate for 1 2 min Note If a heating b
23. his kit was tested according to the user manual cDNA probes were prepared from Control K562 Total RNA PAC0078 Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com ZO md 61 81 n 91 SI ial I ZI 01 6 8 2 9 g 2 1 AUA UATO NOA Xdd ZAION bowsu a94 civawd oyiga unos MHL u4 o ZeVQW ZX4d ZOON L dOIW VISVW Ino Z3 8 9 evHad DIO 197 9 zzwva HANN VNHL DENIS IXIA IXS4 MDIN ZHSW sew o yor o 93 I ISOA Wad IVHd3 poaa ID LBNOIO IXV IOSL I d4O1 1148 ZXVd ZW ddenN ayw 94HVXVW Og IIXOH ava ISW3 OO I usnag VHAV sok 9 ZJNO9AL TAL Nous Olld qq 40 214 AVd CIN qdw ISLV I 2 gTLXd 219930 ZVONA IM IJOL JAL IHONVAS SEN Old 6 4 sow o gayw ovim zaxa zoia 100 Ivona r nue PLUM 6 01 sey SLA 264 INW euro pavi 1 s a ZLX3 DEF pio vudp GINA Zd e G4L I IOL ppewg stu eH esd
24. lock is used tubes must be incubated for a minimum of 2 min at 42 C Prepare the cDNA synthesis mixture for each total RNA sample 6 wl 5X First Strand Buffer 01MDTT 6 ul Probe Mix Reverse Transcriptase 4H HO provided 20 Total volume per RNA sample Mix well by pipetting Incubate the cDNA synthesis mixture at 42 C for 2 min Transfer the mixture to the tube containing the RNA sample from Step B3 and mix well by pipetting Then incubate at 42 C for 2 hr After incubation samples at 75 C for 10 min to inactivate RT Proceed directly to Step D Hybridization or store DIG labeled cDNA probe s at 20 C until use Pre Hybridization Before you start preheat Hybridization Buffer to 52 C Completely dissolve all precipitates in the Hybridization Buffer Prepare sheared salmon testes DNA 1 Heat the sheared salmon testes DNA 10 mg ml at 95 100 C for 5 min then immediately place on ice 5 min We recommend preparing aliquots of 100 ul i e 50 ul per membrane then storing at 20 C until use Place each membrane into a hybridization pouch with the spots facing up Note the orientation of the membranes The correct orientation is shown in the Appendix indicating that the notch is on the top right hand corner Fill the pouch entirely with pre warmed 5 ml deionized H O avoid adding H O directly onto the membrane Then carefully decant water For each membrane prepare pre hybridization solu
25. mone receptor alpha 1 TR alpha 1 gene THRB O17 M26747 Human c erbA mRNA TIAM1 P17 U16296 Human T lymphoma invasion and metastasis inducing TIAM1 protein TIAM1 mRNA TID1 18 _005147 Homo sapiens tumorous imaginal discs Drosophila homolog TID1 mRNA Tim B18 NM 005435 Human Oncogene TIM TIM gene TIMP 3 HUMAN C18 U14394 Human tissue inhibitor of metalloproteinases mRNA TLN D18 NM_006289 Homo sapiens talin TLN mRNA TM4SF1 E18 M90657 Human tumor antigen L6 mRNA TNE F18 X01394 Human tumor necrosis factor TNF b G18 D12614 Human mRNA for lymphotoxin TNF beta TNF c H18 111015 Homo sapiens lymphotoxin beta mRNA TP53BP2 118 U58334 Human Bcl2 p53 binding protein Bbp 53BP2 BBP 53BP2 TPO J18 NM 000460 Human thrombopoietin myeloproliferative leukemia virus oncogene ligand megakaryocyte growth and development factor THPO gene TreORF1 210 K18 X63546 H sapiens mRNA for tre oncogene clone 210 TreORF2 213 L18 X63547 H sapiens mRNA for tre oncogene clone 213 TSC1 M18 AF013168 Homo sapiens hamartin TSC1 mRNA UNPH N18 U20657 Human ubiquitin protease Unph proto oncogene mRNA complete cds USP4 O18 NM 003363 Homo sapiens ubiquitin specific protease 4 proto oncogene USP4 mRNA vav P18 X16316 Human vav oncogene VAV2 C19 NM 003371 Homo sapiens 2 oncogene VAV2 mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110
26. n MENT gene Met c met G11 M60718 Human hepatocyte growth factor gene MKK4 H U17743 Human JNK activating kinase NKK1 mRNA MLH1 H1 U07343 Human DNA mismatch repair protein homolog hMLH1 MN1 J1 NM_002430 Homo sapiens meningioma disrupted in balanced transloca tion 1 MN1 mRNA Mos c mos K11 J00119 Human humos gene homologous to transforming gene of mmsv Mpl c mpl L11 M90102 Human clones 15 39 41 c myeloproliferative leukemia virus type P c mpl P mRNA MRVI1 M11 NM 006069 Homo sapiens murine retrovirus integration site 1 homolog MSH2 N11 U04045 Human hMSH2 mRNA MTCP 1 O11 724459 724460 gene exons 2 to 7 and joined mRNA 12 NM_005962 Homo sapiens MAX interacting protein 1 MXI1 mRNA Myb b myb B12 X13293 Human B myb oncogene Myb c myb C12 M15024 Human Proto oncogene MYBL1 D12 X66087 H sapiens a myb mRNA Myc c myc E12 01841 J00120 Human proto oncogene Myc L myc F12 M19720 Human L myc protein gene Myc n myc G12 Y00664 Human germ line n myc gene NBL1 H12 D28124 Human mRNA for unknown product NE dlg 112 U49089 Human neuroendoctrine dlg NE dlg mRNA NEO1 J12 U61262 Human neogenin mRNA NF1 K12 M89914 Human neurofibromin NF1 gene NF2 L12 NM 000268 Homo sapiens neurofibromatosis type 2 NF2 mRNA NFkB2 M12 X61498 H sapiens mRNA for NF kB subunit NKTR N12 L04288 Homo sapiens cyclophilin related protein mRNA NOC2 O12 AF129812 Homo sapiens candidate tumor suppr
27. o sapiens PR domain zinc finger protein PFM1 mRNA Pim c Pim2 E14 NM 006875 Homo sapiens proto oncogene Pim 2 serine threonine kinase Pim 1 F14 NM 002648 Human pim 1 oncogene PIM1 gene PLA2P 14 AF145020 Homo sapiens phospholipase A2 activating protein PLA2P PP32R1 H14 AF008216 Homo sapiens candidate tumor suppressor pp32r1 PP32R1 PRG1 114 X96438 H sapiens PRG1 gene PRLTS J14 D37965 Human mRNA for PDGF receptor beta like tumor suppressor PTC K14 U43148 Human patched homolog mRNA PTC CP L14 M31213 Human papillary thyroid carcinoma encoded protein mRNA PTCH M14 U43148 Human patched homolog mRNA PTEN N14 U93051 Human putative protein tyrosine phosphatase PTEN gene 1 O14 L41498 Homo sapiens longation factor 1 alpha 1 PTI 1 mRNA PTPG1 P14 569184 PTPG1 tumor suppressor 3 region clone DC1 human colo rectal carcinoma cell line DLD 1 mRNA Partial Mutant 236 nt PVT1 15 X04620 Human lymphoma pvt 1 locus DNA near t 2 8 translocation breakpoint A Raf 1 B15 X04790 Human mRNA for A raf 1 oncogene Raf c rafl C15 X03484 Human mRNA for raf oncogene Raf b D15 NM_004333 Homo sapiens v raf murine sarcoma viral oncogene homolog B1 BRAF Ral A E15 NM 005402 Homo sapiens v ral simian leukemia viral oncogene homolog A ras related RALA mRNA RalB F15 NM 002881 Homo sapiens v ral simian leukemia viral oncogene homolog B ras related GTP binding protein RALB mRNA RAN G15 NM 006325
28. piens requiem homolog hsReq mRNA ING1 A09 AF110645 Human candidate tumor suppressor p33 ING1 homolog gene INGIL B09 NM 001564 Homo sapiens inhibitor of growth 1 like ING1L mRNA IRF 4 C09 U52682 Human lymphocyte specific interferon regulatory factor interferon regulatory factor 4 LSIRF IRF4 mRNA JAW 1 D09 U10485 Human lymphoid restricted membrane protein Jaw1 mRNA Jun c jun E09 J04111 Human c jun proto oncogene JunB F09 X51345 Human jun B mRNA for JUN B protein JunD G09 X51346 Human jun D mRNA for JUN D protein H09 U20770 Human metastasis suppressor mRNA Kit c kit 109 X06182 Human Proto oncogene LAF4 J09 NM 002285 Homo sapiens lymphoid nuclear protein related to AF4 LAF4 Latge T Ag K09 J02400 Simian virus 40 Large T Antigen LATS1 L09 NM_004690 Homo sapiens LATS large tumor suppressor Drosophila homolog 1 LATS1 mRNA LBC M09 NM_006738 Homo sapiens lymphoid blast crisis oncogene LBC mRNA LCK c Lck N09 NM 005356 Homo sapiens lymphocyte specific protein tyrosine kinase LEU1 O09 Y15227 Homo sapiens mRNA for leukemia associated gene 1 Leu2 P09 Y15228 Homo sapiens mRNA for leukemia associated gene 2 Leu5 A10 NM 005798 Homo sapiens candidate tumor suppressor involved in B CLL Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com
29. ss substrate Avoid air bubbles Expose the membrane to Hyperfilm ECL Amersham Cat RPN31144 for 3 10 min at room temperature We suggest trying several different ex posures of varying lengths of time 2 10 min Alternatively use a chemi luminescent imaging system such as Alpha Innotech s FluorChem Imager ot equivalent Interpretation of results For quantitative results we suggest using an chemiluminescent imaging system such as Alpha Innotech s FluorChem or equivalent Be sute to read Section G below to find out how to normalize the genes on the arrays For a quick cutsory analysis of results you may perform the following 1 Place the two x ray images together such that the housekeeping genes aligned 2 Slowly shift one x ray film to the left or right and observe any differences in spot intensity Spots that exhibit differences in signal intensity may indi cate that a gene is upregulated or downregulated however results are not conclusive until you have normalized the genes on the arrays Normalization On each array two housekeeping genes beta actin and ubiquitin have been spotted at different locations on the array Four beta actin spots have been placed on the lower right hand corner for orientation purposes see Appendix Select one housekeeping gene On each array take the average intensity for the housekeeping gene of your choice and use this value as the baseline Normalize each spot with respe
30. tion Add 50 ul of the sheared salmon testes DNA to 5ml of pre warmed Hybridization Buffer After mixing add 5 ml of pre hybridization solution to each pouch con taining the membrane Remove bubbles before sealing the pouch Incubate at 52 C for 22 hr Genemed Biotechnologies Inc Page7 Profiler Array Nonradioactive System IV D E INSTRUCTIONS FOR USE continued Hybridization 1 Heat DIG labeled cDNA probe at 94 C for 5 min Then incubate on ice for 5 min 2 Cutopen hybridization pouch add each DIG labeled cDNA probe directly to the Hybridization Buffer Step C 4 reseal pouch and hybridize over night 12 hours at 52 3 The next day prepare SSC SDS washing solutions as described in the Ap pendix Preheat SSC SDS solutions to 52 C before use Ensure that the SSC SDS solutions are free from all precipitates Shake well before use 4 Cut open hybridization pouch decant cDNA probe buffer and place mem brane into a clear plastic container such as a 200 Ul pipet tip container Quicky tinse each membrane with 10 ml 2X SSC 0 5 SDS and decant Wash twice with 30 ml of prewarmed 2X SSC 0 5 SDS at 52 C for 10 min per wash Then wash twice using 30 ml of prewarmed 0 1X SSC 0 5 SDS at 52 C for 10 min per wash Keep SSC SDS solutions at 52 C during wash steps Detection Note that all incubation and wash steps below should be performed on a rotat ing platform with moderate shaking at room temp 1 Blocking De
31. tudied genes spotted onto a positively charged nylon membrane Star Profiler Array contains genes that play a significant role in biological functions and are corroborated with published literature thus a single experiment using Star Profiler Arrays generates highly informative data In addition each spot contains long oligonucleotides gt 90 nt synthesized from a carefully selected sequence Our selection process eliminates any ho mologous regions among the genes on the array so there is no risk of crosshybridization As a result your labeled cDNA probe will hybridize effi ciently and selectively to specific sequences on the array In addition we offer targeted arrays such as the Oncogene Profiler Array and Apoptosis Profiler Array For a complete list of genes for each array see the Product Certificate Sheet provided with each kit Benefits of the Profiler Array Unlike other macroarrays which often require poly A RNA the Profiler Array has no preference for total or poly A RNA In fact our method provides equivalent results using either total RNA or mRNA so there is no need for special RNA purification procedures Genemed Biotechnologies Inc Page 3 Profiler Array Nonradioactive System I INTRODUCTION continued Sample 1 Sample 2 e g control e g experimental Obtain cell or tissue samples U Uu Isolate Total RNA s ei 1 6 hr AAAA AMAA NA Gv 9 al AAA Z AAA Prep
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