Accelerating Scientific Discovery ÁPlease consider the
Contents
1. IRAK2 IRF 1 IRF3 JUN LTA CD180 LY86 LY96 MAP2K3 MAP2K4 MAPS3K1 MAP3K7 TAB1 MAP4K4 MAPK8 MAPKS8IP3 MYD88 NFKB1 NFKB2 NFKBIA NFKBIL1 NFRKB Accelerating Scientific Discovery Interleukin 6 interferon beta 2 Interleukin 8 Interleukin 1 receptor associated kinase 1 Interleukin 1 receptor associated kinase 2 Interferon regulatory factor 1 Interferon regulatory factor 3 Jun proto oncogene Lymphotoxin alpha TNF superfamily member 1 CD180 molecule Lymphocyte antigen 86 Lymphocyte antigen 96 Mitogen activated protein kinase kinase 3 Mitogen activated protein kinase kinase 4 Mitogen activated protein kinase kinase kinase 1 E3 ubiquitin protein ligase Mitogen activated protein kinase kinase kinase 7 TGF beta activated kinase 1 MAP3K7 binding protein 1 Mitogen activated protein kinase kinase kinase kinase 4 Mitogen activated protein kinase 8 Mitogen activated protein kinase 8 interacting protein 3 Myeloid differentiation primary response gene 88 Nuclear factor of kappa light polypeptide gene enhancer in B cells 1 Nuclear factor of kappa light polypeptide gene enhancer in B cells 2 p49 p100 Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor like 1 Nuclear factor related to kappaB binding protein ey Please consider the environment before printing Tel 12. qPCR MasterMix 1250 ul Diluted cDNA 100 ul nuclease free H2O 1150 ul Total Volume 2500 ul Note save the remainder of the cDNA synthesis reaction and store at 20 C for possible RNA quality analysis in later troubleshooting step 4 Loading the PCR arrays Please select your PCR Array Format for loading instruction 1 1 Carefully remove the PCR Array from its sealed bag 1 2 Dispense Experimental Cocktail to PCR Array Loading Reservoir to assist in loading optional 1 3 Add 25 ul of the Experimental cocktail to each well of the PCR Array preferably from a reservoir with an eight or twelve channel pipettor 5 Performing realtime PCR detection Attention Users of Bio Rad and Eppendorf Realtime instruments prior to initiating the run make sure your instrument has been calibrated for using clear sticky film Note follow the manufacturer s instruction for the proper operation and maintenance of your realtime instrument 5 1 Carefully and tightly seal the PCR Array with the optical thin adhesive film 5 2 Centrifuge the plate for 1 full minute at 4 C at 1000g to remove bubbles Visually inspect the plate from underneath of the plate to ensure no bubbles are present in each well 5 3 Place the plate on ice while setting up the PCR cycler program below 5 4 Place the plate in your realtime thermal cycler if recommended by your instrument s user manual use a compression pad with the optical film sealed plate f
3. alpha 1 Interferon beta 1 fibroblast Interferon gamma Inhibitor of kappa light polypeptide gene enhancer in B cells kinase beta Interleukin 10 Interleukin 12A natural killer cell stimulatory factor 1 cytotoxic lymphocyte maturation factor 1 p35 Interleukin 1 alpha Interleukin 1 beta Interleukin 2 Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme C5 C6 C7 C8 c9 C10 C11 C12 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E1 E2 E3 E4 E5 NM_000600 3 NM_000584 3 NM_001569 3 NM_001025243 1 NM_001025242 1 NM_001570 3 NM_002198 2 NM_001571 5 NM_001197128 1 NM_001197127 1 NM_001197125 1 NM_001197126 1 NM_001197124 1 NM_001197123 1 NM_001197122 1 NM_002228 3 NM_000595 3 NM_001159740 2 NM_005582 2 NM_004271 3 NM_015364 4 NM_001195797 1 NM_002756 4 NM_145109 2 NM_003010 2 NM_005921 1 NM_003188 3 NM_145333 2 NM_145332 2 NM_145331 2 NM_006116 2 NM_153497 2 NM_004834 4 NM_145687 3 NM_145686 3 NM_001242560 1 NM_001242559 1 NM_002750 2 NM_139049 1 NM_139047 1 NM_139046 1 NM_015133 3 NM_001040439 1 NM_002468 4 NM_001172568 1 NM_001172569 1 NM_001172567 1 NM_001172566 1 NM_003998 3 NM_001165412 1 NM_002502 4 NM_001077494 2 NM_001261403 1 NM_020529 2 NM_005007 3 NM_001144963 1 NM_001144962 1 NM_001144961 1 NM_006165 3 NM_001143835 1 IL6 IL8 IRAK1
4. 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme E6 E7 E8 E9 E10 E11 E12 F1 F2 F3 F4 F5 F6 F7 F8 F8 F9 F10 F11 F12 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 NM_003298 3 NM_020651 3 NM_005036 4 NM_001001928 2 NM_003690 4 NM_001139518 1 NM_001139517 1 NM_000963 2 NM_002908 2 NM_021975 3 NM_001145138 1 NM_001243985 1 NM_001243984 1 NM_003821 5 NM_015077 2 NM_021805 2 NM_001135054 1 NM_001135053 1 NM_016581 4 NM_001 142465 2 NM_001142464 2 NM_001243204 1 NM_013254 3 NM_021649 6 NM_181836 5 NM_001164469 2 NM_001164468 2 NM_001039661 1 NM_148910 2 NM_003263 3 NM_030956 3 NM_001017388 2 NM_001195108 1 NM_001195107 1 NM_001195106 1 NM_003264 3 NM_003265 2 NM_138554 4 NM_003266 3 NM_138557 2 NM_003268 5 NM_006068 4 NM_016562 3 NM_138636 4 NM_017442 3 NM_000594 3 NM_001065 3 NM_019009 3 NM_004620 3 NM_145803 2 NM_182919 3 NM_003348 3 NM_021988 5 NM_199129 2 NM_199203 2 NM_199144 2 NR2C2 PELI1 PPARA PRKRA PTGS2 REL RELA RIPK2 SARM1 SIGIRR ECSIT TBK1 TICAM2 TIRAP TLR1 TLR10 TLR2 TLRS TLR4 TLRS TLR6 TLR7 TLR8 TLR9 TNF TNFRSF1A TOLLIP TRAF6 TICAM1 UBE2N UBE2V1 Accelerating Scientific Discovery Nuclear receptor subfamily 2 group C member 2 Pellino E3 ubiquitin protein ligase 1 Peroxisome proliferator activated receptor alpha Protein kinase interferon indu
5. ECSIT TBK1 TICAM2 TIRAP TLR1 TLR10 TLR2 TLR3 TLR4 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 TLR5 TLR6 TLR7 TLR8 TLR9 TNF TNFRSF1A TOLLIP TRAF6 TICAM1 UBE2N UBE2V1 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 RIPK3 RGS2 RPL13A B2M HGD1 HGD2 GAPDH GAPDH ACTB ACTB TUBA1B HPRT1 S Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme Order Information We have two formats of 2x Elite qPCR MasterMix for different type of the realtime thermal cyclers Accelerating Scientific Discovery e Format A is suitable for use with the real time thermal cyclers that do not require a reference dye Bio Rad models CFX96 CFX384 Bio Rad MJ Research models Chromo4 DNA Engine Bio Rad models iCycler iQ5 MyiQ MyiQ2 Opticon 2 Roche LightCycler 480 96 well Human TLR Pathways qPCR Human TLR Pathways qPCR GEOR Array Formatie Array trial size Cat GA R204A1 Array Cat GA R204A 96 Well Plate Containing Dried Assays Part R204 120 2 plates 12 plates Adhesive Film Part GA 005 2 pieces 12 pieces 2x Elite qPCR MasterMix HotStart Tag dNTP EvaGreen Dye 2x 1 25 ml 12x 1 25 ml Part GA 135 e Format B is suitable for use with the following real time thermal cyclers Applied Biosystems models 5700 7300 7500 Standard and Fast 7700 7900HT Standard and Fast StepOnePlus ViiA7 Standard and Fast Eppend
6. NM_002923 3 NM_012423 3 NM_001270491 NM_004048 2 BSG 0001 BSG 0002 NM_002046 4 NM_001256799 1 NM_002046 4 NM_001256799 1 NM_001101 3 NM_001101 3 NM_006082 2 NM_000194 2 RIPK3 RGS2 RPL13A B2M HGD1 HGD2 GAPDH GAPDH 1 ACTB ACTB 1 Tubaib HPRT1 Accelerating Scientific Discovery Receptor interacting serine threonine kinase 3 Regulator of G protein signaling 2 24kDa Ribosomal protein L13a Beta 2 microglobulin Human Genomic DNA Contamination Human Genomic DNA Contamination Glyceraldehyde 3 phosphate dehydrogenase Glyceraldehyde 3 phosphate dehydrogenase Actin beta Actin beta Homo sapiens tubulin alpha 1b TUBA1B Hypoxanthine phosphoribosyltransferase 1 ey Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com
7. lt GEnzyme Human TLR Pathways qPCR Array Catalogue GA R204A GA R204B Accelerating Scientific Discovery Description Toll like receptors TLRs are single membrane spanning non catalytic receptors that activate immune cell responses upon microbes invasion This Human TLR Pathways Array is designed to profile the expression of 88 genes for the receptors in the TLR family as well as the adapter proteins and kinases that mediate TLR signaling Our PCR Array plates are pre coated with EvaGreen optimized primer assays for a thoroughly researched panel of relevant pathway or disease focused genes Our unique high quality primer design and master mix formulation enable the PCR Array to amplify 96 different gene specific products simultaneously under uniform cycling conditions All primer sets designed by our expertise scientists are able to amplify the alternative splice variants of corresponding target genes A few additional house keeping genes are used as positive controls Features e High Sensitivity cDNA made from as little as 1 ng or as much as 5 ug of total RNA per array plate provides greater than 85 percent present call rates e High Reproducibility the system has replicate correlation coefficients gt 0 99 which means that experimental samples can be reliably compared across plates and runs e High Specificity the combination of EvaGreen primers and 2x Elite qPCR MasterMix guarantees a single product of the predicted
8. _176892 1 NM_001278 3 NM_014358 2 NM_000758 3 NM_000759 3 NM_001178147 1 NM_172220 2 NM_172219 2 NM_001565 3 NM_002759 3 NM_001135652 2 NM_001135651 2 NM_005229 4 NM_001114123 2 NM_001257168 1 NM_003824 3 NM_005252 3 NM_002128 4 NM_005343 2 NM_176795 3 NM_001130442 1 NM_005345 5 NM_002156 4 NM_199440 1 NM_024013 2 NM_002176 2 NM_000619 2 NM_001556 2 NM_001190720 2 NM_001242778 1 NM_000572 2 NM_000882 3 NM_000575 3 NM_000576 2 NM_000586 3 Symbol BTK CASP8 CCL2 CD14 CD80 CD86 CHUK CLEC4E CSF2 CSF3 CXCL10 EIF2AK2 ELK1 FADD FOS HMGB1 HRAS HSPA1A HSPD1 IFNA1 IFNB1 IFNG IKBKB IL10 IL12A IL1A IL1B IL2 Accelerating Scientific Discovery Name Bruton agammaglobulinemia tyrosine kinase Caspase 8 apoptosis related cysteine peptidase Chemokine C C motif ligand 2 CD14 molecule CD80 molecule CD86 molecule Conserved helix loop helix ubiquitous kinase C type lectin domain family 4 member E Colony stimulating factor 2 granulocyte macrophage Colony stimulating factor 3 granulocyte Chemokine C X C motif ligand 10 Eukaryotic translation initiation factor 2 alpha kinase 2 ELK1 member of ETS oncogene family Fas TNFRSF6 associated via death domain FBJ murine osteosarcoma viral oncogene homolog High mobility group box 1 V Ha ras Harvey rat sarcoma viral oncogene homolog Heat shock 70kDa protein 1A Heat shock 60kDa protein 1 chaperonin Interferon
9. cible double stranded RNA dependent activator Prostaglandin endoperoxide synthase 2 prostaglandin G H synthase and cyclooxygenase V rel reticuloendotheliosis viral oncogene homolog avian V rel reticuloendotheliosis viral oncogene homolog A avian Receptor interacting serine threonine kinase 2 Sterile alpha and TIR motif containing 1 Single immunoglobulin and toll interleukin 1 receptor TIR domain ECSIT homolog Drosophila TTANK binding kinase 1 Toll like receptor adaptor molecule 2 Toll interleukin 1 receptor TIR domain containing adaptor protein Toll like receptor 1 Toll like receptor 10 Toll like receptor 2 Toll like receptor 3 Toll like receptor 4 Toll like receptor 5 Toll like receptor 6 Toll like receptor 7 Toll like receptor 8 Toll like receptor 9 Tumor necrosis factor Tumor necrosis factor receptor superfamily member 1A Toll interacting protein TNF receptor associated factor 6 E3 ubiquitin protein ligase Toll like receptor adaptor molecule 1 Ubiquitin conjugating enzyme E2N Ubiquitin conjugating enzyme E2 variant 1 ey Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 NM_001032288 2 NM 022442 5 NM_001162505 1 NM_001257395 1 NM_001257394 1 NM_001257393 1 NM_001257399 1 NM_001257398 1 NM_001257397 1 NM_001257396 1 NM_006871 3
10. cision of pipetting determines the consistency of the results Make sure that all the micro pipettors used are calibrated and not to introduce any bubbles into the wells of the PCR Array 5 DEPC treated H2O should NOT be used Use high quality nuclease free H2O Check with the supplier if not sure whether your RNase DNase free water has been treated with DEPC 6 Exam the quality of your sample RNA before starting the experiment If precipitates are present in eEnzyme 2x Elite qPCR MastMix tubes please contact a technical application scientist at 1 800 919 0755 or info eenzyme com 8 Regarding the concern of genomic DNA contamination our arrays are designed to skip at least one intron so that traces of contaminated genomic DNA in the sample if there is any will not be amplified In addition each pair of primers are designed to have 60 C 1 annealing temperature which guarantees that large sized genomic DNA if any cannot be amplified Accelerating Scientific Discovery A 9 N Workflow and Protocols 1 Make cDNA from your sample RNA refer to your reverse transcription kit manual not included in the array kit 2 Thaw 2x Elite qPCR MasterMix on ice vortex and briefly spin down 3 Mix all following components in a tray for multi channel pipetting Carefully pipette precise 25 ul reaction mix to each of the 96 wells Change pipet tips following each addition to avoid any cross contamination 2x Elite
11. ible amplification Set the instrument to use the readings from cycle number two 2 through two 2 cycles before the earliest visible amplification but no more than cycle 15 The earliest amplification usually will be visible between cycles 14 and 18 ii Manually define the threshold value by using the log view of the amplification plots and place it above the background signal but within the lower one third to lower one half of the linear phase of the amplification plot Important ensure that the thresholds are the same across all PCR Array runs in the same analysis The absolute position of the threshold is less critical than its consistent position across arrays When the quality of the RNA sample adequately controlled the cycling program executed properly and the thresholds defined correctly the value of Ct should be 20 2 cross all of your arrays or samples jii Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with the PCR Array Data Analysis Template Excel 6 Recommended Quality Control Dissociation Melting Curve For instrument specific melt curve analysis settings please refer to the corresponding instrument Setup Guide Note If you decide not to obtain the dissociation curve immediately save the plates in aluminum foil at 20 C as is in case you need to do this operation at a later time for troubleshooting When ready simply warm the plate to room temperature place it into
12. orf Mastercycler ep realplex models 2 2S 4 4S Stratagene models Mx3000P Mx3005P Mx4000 Takara TP 800 Human TLR Pathways qPCR Human TLR Pathways qPCR ROR Array Formar E Array trial size Cat GA R204B1 Array Cat GA R204B 96 Well Plate Containing Dried Assays Part R204 120 2 plates 12 plates Adhesive Film Part GA 005 2 pieces 12 pieces 2x Elite qPCR MasterMix HotStart Tag dNTP EvaGreen Dye ROX Passive Reference Dye Part GA 245 2x 1 25 ml 12x 1 25 ml Storage Keep in freezer 20 C and avoid exposure to light Materials Required But Not Included e The Reverse transcription reagents for making the cDNA from your prepared total RNA are not included in the array kit Protocol and reagents from Invitrogen and Qiagen for reverse transcription have been tested and worked well along with this kit e High quality nuclease free water Do not use DEPC treated water e Low EDTA TE buffer 0 1 mM EDTA Important Notes before Use 1 Please read through this entire protocol before beginning your experiment 2 The use of eEnzyme 2x Elite qPCR MastMix included is critical for obtaining the most accurate results from the PCR Array my Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme Make sure you have the correct PCR array plate format for your realtime PCR instrument to avoid damage The accuracy and pre
13. ormats Note PCR Arrays containing experimental cocktail may be store at 20 C wrapped in aluminum foil for up to one week until ready to run 5 5 Enter and run the appropriate program for your realtime instrument We provide a file to help customs easy to load software for both ABI and Bio Rad realtime PCR instruments A Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com lt GEnzyme Use a Two step cycling program for the following instrumentation ABI 5700 7000 7300 7500 7700 7900HT Accelerating Scientific Discovery StepOnePlus Bio Rad icycler IQ5 MyiQ MyiQ2 CFX96 CF384 49 Eplpendorf Mastercycler ep realplex 2 2s 4 4S Stratagene Mx3000p Mx3005p Mx4000p Attention Bio Rad CFx96 amp CF384 users adjust the ramp rate to 1 C sec 5 6 Calculate the threshold cycle Ct for each well using the instrument s software Note for Roche Light Cycler 480 Users there are two options available to analyze your data Use the second derivate max setting and there is no need to set a threshold i To define the Baseline Choose the Automated Baseline option if your instrument has the Adaptive Baseline Function check with instrument manual or manufacturer if unsure If it does not have the adaptive baseline function you will need to set the baseline manually Use the Linear View of the amplification plots to determine the earliest vis
14. size from every reaction without secondary products such as primer dimers Controls are also included for monitoring genomic DNA contamination RNA quality and general PCR performance e Easy to Use simple experiment workflow and easy to use Excel based template for data analysis The analysis is based on the AAC method with normalization of the raw data to either housekeeping genes or an external RNA control This PCR Array is compatible with but not limited to all ABI Bio Rad Eppendorf QIAGEN Roche and Stratagene instruments Kit Components e 2x Elite qPCR MasterMix HotStart Tag dNTP EvaGreen Dye ROX Passive Reference Dye included for format B Adhesive films 1 piece each plate e Manual and PCR Data Analysis Tool one CD included 96 well plate array see the table below for the genes included A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 BTK CASP8 CCL2 CD14 CD80 CD86 CHUK CLEC4E CSF2 CSF3 CXCL10 EIF2ZAK2 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 ELK1 FADD FOS HMGB1 HRAS HSPA1A HSPD1 IFNA1 IFNB1 IFNG IKBKB IL10 C1 C2 C3 C4 C5 C6 C7 C8 cg C10 C11 C12 IL12A ILIA IL1B IL2 IL6 IL8 IRAK1 IRAK2 IRF1 IRF3 JUN LTA D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 CD180 LY86 LY96 MAP2K3 MAP2K4 MAP3K1 MAP3K7 TAB1 MAP4K4 MAPK8 MAPK8IP3 MYD88 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 NFKB1 NFKB2 NFKBIA NFKBIL1 NFRKB NR2C2 PELIT PPARA PRKRA PTGS2 REL RELA F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 RIPK2 SARM1 SIGIRR
15. your realtime instrument and run the melting program described above i Be sure to visually inspect the plate after the run for any sign of evaporation from any of the wells If evaporation is observed make a note of which wells so that you may qualify your data analysis appropriately ii Do not open any previously run and stored PCR Array plate Removing the adhesive film to see if PCR product is evaporated during PCR process ili Run a melting curve program immediately after the above cycling program and generate a first derivative dissociation curve for each well in the entire plate using your instrument s software No more than one peak should appear in each reaction at temperatures greater than 80 C If your instrument does not have a default melting curve program run the following program instead 95 C 1min 65 C 2min Optics off 65 C to 95 C at 2 C min Optics ON Please consider the environment before printing Tel 1 240 683 5851 e Fax 1 240 683 5852 e info eEnzyme com www eEnzyme com Enzyme Gene Information Position GeneBank A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 C1 C2 C3 C4 NM_000061 2 NM_001228 4 NM_001080125 1 NM_001080124 1 NM_033358 3 NM_033356 3 NM_033355 3 NM_002982 3 NM_000591 3 NM_001040021 2 NM_001174105 1 NM_001174104 1 NM_005191 3 NM_006889 4 NM_175862 4 NM_001206925 1 NM_001206924 1 NM
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