Multiplex 96×96 - Olink Bioscience
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1. Olink Bioscience O L N K Dag Hammarskj lds v 52B SE 752 37 Uppsala Sweden BIOSCIENCE www olink com 0935 v 1 4 2014 05 242. on the left side of the chip in z OO P the same manner as described o LLT in steps 1 3 for Sample Plate gt e c M OO NM 1 XD Primer Plate Primers Samples 18 Multiplex 9 9 Appendix 2 TISSUE LYSATES Proseek Multiplex 9 15 compatible with human tissue lysates Two different lysis buffers and different tissues such as lung tissue muscle tissue endometrial tissue and skin melanoma tissue have been selected Optimization of lysis buffer may be necessary depending on tissue type Make sure you have equal amount of tissue In each sample Determine the total protein concentration in each sample and standardize by diluting high level samples Samples standardized to approximately 0 5 1 mg mL based on protein determination carried out using BCA Protein Assay Lowry show good result with Proseek Multiplex 98 9 Specific tissues could have very high expression of certain proteins and further dilution may be necessary for the Proseek Multiplex assay e g 1 50 or 1 100 LYSIS BUFFER 1 10x RIPA buffer 50mM Tris HCl pH 74 150 mM NaCl 1 mM EDTA 1 Triton X 100 0 1 Sodium deoxycholate Add fresh to 1x buffer gt Serine protease inhibitor Phenylmethylsulfony fluoride PMSF to 1 mM Protease Inhibitor cocktail final conc 10 4 mM AEBSF 8 uM Aprotinin 0 2 mM Leupeptin 0 4 mM Bestatin 0 15 mM Pepstatin 0 14 mM E 64 For protein determination using BCA Lowry method dilute s
3. 12 A 1 m O w gt 1 m O w gt 200008 99999990 239999999 9999999 e Primer Plate Sample Plate oe eC ee ee Primers Samples Fig 3 Loading of samples and primers to the 96 96 Dynamic Array IFC Note The chip should be oriented so that the cut corner of the chip is placed Remove any visible bubbles e g with a pipette tip or syringe needle your upper left side and change between each well Load the chip in the Fluidigm IFC Controller HX according to manufacturer s instructions Run the Olink Protein Expression 96x96 Program in the Fluidigm Biomark Reader according to manufacturer s instructions See 5 3 for detailed instructions on the Olink Protein Expression 96x96 Program Proseek Multiplex 9 6 15 6 Results and data analysis 6 1 EXPORT THE DATA Verify the BioMark run using the Fluidigm Real Time PCR analysis software according to manufacturer s instructions Export your data to a spreadsheet see Olink Wizard for GenEx User Guide for a detailed instruction 6 2 DATA UNITS The Proseek assay generates Cq values To even out variation between runs and within run the data should be normalized using the Extension Control Interplate Control and a correction factor The data used for further statistical analysis is in Normalized Protein Expression NPX units on log2 scale where a high value corresponds to high protein concentr
4. normalization and compensates for the variation between samples in the Extension and Detection steps of the assay The Detection Control is a synthetic oligonucleotide that monitors the Detection step This control is not affected during Incubation or Extension three replicates of Interplate Control are used for normalization and compensates for possible variation between runs The three replicates of Negative Control are used to calculate the lower limit of detection LOD for each protein Proseek Multiplex 4 Assay considerations 4 1 SAFETY Follow general laboratory safety procedure such as using gloves safety goggles and protective clothing when performing the experiments Handle and dispose of hazardous sample material according to local regulations 4 2 PCR TECHNOLOGY PCR technology is sensitive to contaminations perform the Detection step in a post PCR room separate from the room where the Incubation and Extension steps to step 19 page 13 are performed Maintain and calibrate your PCR and BioMark instruments regularly 4 3 PIPETTING TECHNIQUES It is advisable to use a multi channel pipette in the reagent transfer steps Use filter pipette tips to avoid contamination Change pipette tips between all sample and reagent transfer steps to avoid cross contamination Maintain and calibrate your pipettes regularly 4 4 SAMPLE PREPARATION To reduce sample handling time during the experiment samples can b
5. reporter sequences by proximity samples extension DETECTION Quantify each biomarker s DNA reporter using high throughput real time PCR instrument Fig 1 Assay procedure Proseek Multiplex 99 5 5 6 3 Reagents and equipment 3 1 REAGENTS SUPPLIED Each Proseek Multiplex 78 98 kit contains reagents for 96 wells sufficient for 90 samples and 6 controls The reagents are supplied in three individual boxes Storage temperature and expiry date for the components are stated on the outer label of each box 3 1 1 PROSEEK MULTIPLEX PROBE STORE AT 4 C d Incubation Solution Contains components needed for the incubation reaction A probes Contains 96 antibody probes labeled with A oligos B probes Contains 96 antibody probes labeled with B oligos 3 1 2 PROSEEK MULTIPLEX DETECTION KIT 995 STORE AT 20 C 4 PEA Solution Contains components needed for the extension reaction PEA Enzyme For extension of A and B probes which are bound to their target PCR Polymerase For pre amplification of the extension product created by the PEA enzyme Detection Solution Contains components needed for the detection reaction Detection Enzyme For GPCR amplification Primer Plate 96 well plate with ready to use primers for amplification of extension product 3 1 3 PROSEEK MULTIPLEX CONTROLS STORE AT 20 C 4 Interplate Control For normalization between runs Negative Control F
6. the 8 well strip to the bottom of each well of a 96 well plate by using reverse pipetting Do not change pipette tips Name this plate Incubation Plate that all wells are properly sealed especially around the edges to avoid evaporation of samples Spin down the content at 150 x g 1 min at room temperature Incubate the ncubation Plate overnight at 2 C to 8 C Proseek Multiplex Note Pipette from the uppermost part of the Incubation mix to prevent liquid from sticking to the outside of the pipette tip Day 2 12 13 14 15 16 17 18 19 20 21 22 Turn on your thermal cycler and activate the heated lid function 2 PEN EXTENSION 8 Thaw the PEA Solution vortex and spin briefly Prepare the following Extension mix in a centrifuge tube Use a freezing block when removing the PEA Enzyme and the PCR Polymerase from 20 C and spin down the content briefly before pipetting the enzymes into the mix Extension mix per 96 well plate uL High Purity Water 9385 PEA Solution 1100 PEA Enzyme b5 PCR Polymerase 22 Total 10 562 Vortex the Extension mix Bring the ncubation Plate to room temperature Spin down the content at 150 x g 1 min at room temperature Pour the Extension mix into a multi channel pipette reservoir Carefully remove the plastic adhesive film from the Incubation Plate Start a timer set for 5 min and transfer 96 uL of Extension mix to each Not
7. 99999 Negative Control Interplate Control Samples Fig 2 Plate layout Proseek Multiplex 5 3 PEA PROGRAM Create a PEA program on the thermal cycler with the following conditions Enable the heated lid function Extension 50 C 20 min Hot start 95 C 5 min PCR Cycle 95 C 30 s 54 C 1 min x17 60 C 1 min Maintain the reaction at 10 C oo hold 5 4 OLINK PROTEIN EXPRESSION 96x96 PROGRAM Program the Fluidigm BioMark System with the following steps Name the program Olink Protein Expression 96x96 program Thermal mix 50 C 120s 70 C 1800 s 25 C 600 s Hot Start 95 C 300 s PCR Cycle 95 C 15s x40 60 C 60s Verify correct settings Application Gene Expression Passive Reference 5 Carboxy x Rhodamine abbreviation ROX in Fluidigm software Assay single probe Probes FAM MGB 5 5 FLUIDIGM INSTRUCTIONS For information on the Fluidigm IFC Controller HX and Fluidigm BioMark System please read through the following User Guides www fluidigm com gt FluidigmQ IFC Controller User Guide PN 68000112 FluidigmQ Real Time PCR Analysis User Guide PN 68000088 FluidigmQ Data Collection Software User Guide PN 68000127 Proseek Multiplex 11 5 6 PROSEEK MULTIPLEX PROTOCOL Before starting Please read the entire Proseek Multiplex 9 6 protocol Decide how many samples you will include in the experiment and the number of replicates Use the 96 well plate template in Figu
8. PENDIX T 17 19 Tissue Lysates 19 EUdgpdge 19 Lysis buffer 2 tcn bases tete iust S eL LO Ete E 19 Proseek Multiplex 3 4 1 Introduction Proseek Multiplex from Olink Bioscience is a diverse product line of reagents for scalable immunoassays enabling simultaneous measurement of 92 protein biomarkers in 1 uL sample volume The Proseek platform Is designed for ease of use and offers enhanced analytical performance analysis of complex matrices as well as improvement in assay throughput over conventional immunoassays To get you started Proseek Multiplex 9 9 reagents come as a convenient all in one kit format with an optimized protocol Proseek Multiplex 2 Principle of the assay 2 1 TECHNOLOGY AND ASSAY FORMAT The Proseek reagents are based on PEA a Proximity Extension Assay technology in which 96 oligonucleotide labeled antibody pairs are allowed to bind to their respective protein targets in the sample A PCR reporter sequence is formed by a proximity dependent DNA polymerization event and is subsequently detected and quantified using real time PCR The assay is performed in a homogeneous 96 well format with no need for washing steps Proseek Multiplex assay procedure employs three core steps INCUBATION EXTENSION Allow the 96 antibody probe pairs to Create and pre amplify 96 unique bind to their respective proteins in your DNA
9. Proseek Multiplex OLINK BIOSCIENCE TECHNICAL SUPPORT For technical support please contact us at support olink com or 46 18 444 3970 Table of content 1 INTRODUCTION octets olea tedere 4 2 PRINCIPLE OF THE ASSAY 5 2 1 Technology and assay format csset 5 3 REAGENTS AND EQUIPMENT en ead p od Dt diete 6 3 1 Reagents supplied crt eee b 6 3 2 Required consumables not supplied nnen 7 3 3 Required equipment not supplied nnen 7 3 4 Software for analysis neee 8 3 5 DownloadS concubitu arcet bant stan apart ete 8 3 6 Technical controls annette 8 4 ASSAY CONSIDERATIONS 9 eneen ME ud iE 9 42 PCR Technology enden 9 4 3 Pipetting techniques 9 4 4 Sample preparation ssec 9 45 Sample material nrden abe eet bet Aaen 9 5 CSOBIDIUFI 10 5 1 Experimental design nennen eet 10 5 2 Plate layout znne eeb 10 5 3 PEA Program zor tenen Rat mind erbaut 11 5 4 Olink Protein Expression 96x96 program 11 5 5 Fluidigm instructions oasis ret ia titii tetti 11 5 6 Proseek Multiplex 99 protocol eneen 12 6 RESULTS AND DATA ANALYSIS ebat bienes ibi edt 16 6 1 Export the data oreet md sitom tab aite aeo euin ete 16 0 2 RN INIT 16 6 3 Olink Wizard for GenEx Software nennen 16 PETRI NEN T 16 AP
10. amples 1 5 in PBS LYSIS BUFFER 2 Bio Plex Cell lysis kit cat number 171 304011 Bio Rad gt Add fresh to buffer Factors 1 and 2 from the kit Protease inhibitor cocktail from Roche cat number 11 836153001 For questions please contact support olink com Proseek Multiplex 9 e 19 For Research Use Only Not for Use in Diagnostic Procedures This product includes a license for non commercial use of Proseek products Commercial users may require additional licenses Please contact Olink AB for details There are no warranties expressed or implied which extend beyond this description Olink AB is not liable for property damage personal injury or economic loss caused by this product The following trademarks are owned by Olink AB Olink Olink Bioscience Proseek Duolink and PLA This product is covered by several patents and patent applications including US 6 511 809 US 7306 904 US 5 665 539 and related US and foreign patents This product is sold under license from PHRI Properties Inc and may be used under PHRI Properties patent rights outside the field of human in vitro diagnostics Components in the Proseek Multiplex Probe Kit utilise Lightning Link technology and are provided under license from Innova Biosciences Fluidigm and BioMark are trademarks or registered trademarks of Fluidigm Corporation All third party trademarks are the property of their respective owners Copyright 2014 Olink AB
11. ation For calculating Coefficient of Variation 96 CV between replicate samples you need to use linear values Convert your NPX values to linear values by using this formula 2 NPX For normalization of your data please use the Olink Wizard for GenEx or download the DataPreprocessing xlsx file corresponding to your panel at www olink com 6 3 OLINK WIZARD FOR GENEX SOFTWARE Each Proseek Multiplex 9 6 experiment will generate 9216 data points To facilitate data analysis we recommend the Olink Wizard plugin for GenEx GenEx is a multivariate statistical analysis software developed by MultiD Analyses AB www multid se The Olink Wizard for GenEx will guide you through data qualification steps and provide you with Normalized Protein Expression NPX values for further statistical analysis The GenEx software offers a variety of statistical analysis tools such as hierarchical clustering methods principal components analysis ANOVA tests and more Different visualization tools including scatter plot box and whisker plot or bar graph allow you to rapidly identify major differences across samples and provide you with images for data presentations For further information about data analysis please contact Olink at support olink com 7 References 1 Lundberg M et al Homogeneous antibody based proximity extension assays provide sensitive and specific detection of low abundant proteins in human blood Nucleic Acid Res 6 June 2011 d
12. e Perform steps 18 20 well of the ncubation Plate by using reverse pipetting Do not change within 5 minutes pipette tips place the tips against the upper parts of the well wall Make sure the tips never come in contact with the contents of the wells Add a new plastic adhesive film to the Incubation Plate It is important that all wells are properly sealed especially around the edges to avoid evaporation of samples Vortex gently and spin down the content at 150 x g 1 min at room temperature After the 5 min place the ncubation Plate in the thermal cycler and Note f your thermal cycler run the PEA program see section 5 2 for details The PEA program requires a silicon cover for takes approximately 1 h 40 min plates covered with plastic film please use one to avoid evaporation Continue with the Detection step or store the Incubation Plate for up to one week at 4 C Proseek Multiplex 13 DETECTION i Q 23 Prepare and prime a 96 96 Dynamic Array IFC according to the manufacturer s instructions 24 Thaw the Primer Plate vortex and spin at 150 x g 1 min at room temperature 25 Thaw the Detection Solution vortex and spin briefly Prepare the following Detection mix in a microcentrifuge tube Use a freezing block for the Detection Enzyme and PCR Polymerase and spin down the content briefly before pipetting the enzymes into the mix Detection mix per 96 well plate uL Detection So
13. e aliquoted in 8 well strips or 96 well plate prior to the start of the experiment 4 5 SAMPLE MATERIAL Proseek Multiplex 9 6 has been validated on EDTA plasma all panels and serum Oncology and Inflammation samples A range of additional sample types are compatible with the technology such as citrate plasma heparin plasma tissue and cell lysates and saliva Different sample matrices can affect the detection of specific proteins For more information on sample material please see the data package corresponding to each panel Guidelines on tissue lysate buffers are available in Appendix 2 For questions please contact supportQolink com Proseek Multiplex 9 10 5 Assay protocol 5 1 EXPERIMENTAL DESIGN It is Important to plan your experimental design properly to get the data you need to answer your questions Decide how many samples replicates and controls you will need to get the data you want Consult a statistician to be on the safe side prior to running your study 5 2 PLATE LAYOUT Prior to running the Proseek Multiplex assay plan the distribution of samples across the plate It is important to place the Negative Control and the Interplate Control in the last 6 wells according to Figure 2 Proseek Multiplex 999 kit is designed for 90 samples three replicates of Negative Control and three replicates of Interplate Control For analysis of less than 90 samples please pipette replicates of selected samples 23
14. lution 550 0 High Purity Water 230 0 Detection Enzyme 78 PCR Polymerase 3 1 Total 790 9 26 Vortex the Detection mix and spin briefly Transfer 95 uL of the Detection mix per well to an 8 well strip 27 Use a multi channel pipette to transfer 72 uL of Detection mix to each well of a new 96 well plate by reverse pipetting Name this plate Sample Plate 28 Remove the ncubation Plate from the thermal cycler vortex and spin down the contents 29 Carefully remove the plastic film and transfer 2 8 uL from each well of the ncubation Plate to the Sample Plate 30 Seal the Sample Plate with a new plastic adhesive film vortex and spin at 150 x g 1 min at room temperature 14 Proseek Multiplex 9 9 31 32 33 34 35 Transfer 5 uL from each well of the Sample Plate to the primed 96 96 Note For steps 31 and 32 Dynamic Array IFC by using reverse pipetting Change pipette tips make sure not to leave any after each sample Samples are loaded into their respective inlets on inlets empty on the chip the right side of the chip according to Figure 3 See Appendix 1 for a detailed instruction on sample loading Gently remove the Primer Plate aluminum sealing to avoid contamination between wells Transfer 5 uL from each well of the Primer Plate into the inlets on the left side of the chip according to Figure 3 by reverse pipetting Change pipette tips after each transfer 8 9 10 1 12 A a 5 6 7 8 9 10 1
15. oi 10 1093 nar gkr424 2 Assarsson E et al Homogenous 96 Plex PEA Immunoassay Exhibiting High Sensitivity Specificity and Excellent Scalability PLOS One 6 April 2014 9 4 e95192 16 Proseek Multiplex 6 Appendix 1 Load samples to the right and primers to the left on the 96 96 Dynamic Array IFC PAP 000000 eee es ee 6000 Sample Plate P P P P P Pe P P eee 16 Primer Plate Samples Primers 17 Proseek Multiplex 9 9e 1 Use reverse pipetting Transfer 5 uL from each well n OX X Y XX Y Y X LL in position 1 A H marked in 9 green to inlets in the first column on the right side of s In z the chip green When using x lO an eight channel pipette 2 19 every other inlet will be filled e Sample Plate according to the image FE ee 2 Transfer 5 uL from each well in position 2 A H blue to the second column of inlets blue e l gt according to image Continue use with columns 3 6 99 4 9 Nr Sample Plate Primers Samples 3 Transfer 5 uL from each well 5 in position 7 A H red to inlets ROO X in the first column on the right NNUS gt side of the chip red start on the second row according to image Continue with columns C 9 12 x a l s Sample Plate Primers Samples 4 Transfer 5 uL from each well fp in the Primer Plate to the inlets ox xxxxxoo
16. or determination of background levels Incubation Stabilizer For stabilization of the incubation reaction Proseek Multiplex 3 2 REQUIRED CONSUMABLES NOT SUPPLIED d d d Pipette tips filter is required Microcentrifuge tubes 1 1 5 mL Centrifuge tube 11 mL 8 well strips with lids 96 well PCR plate 0 2 mL Multi channel pipette reservoir Adhesive plastic film heat resistant High purity water sterile filtered or similar 96 96 Dynamic Array Integrated Fluidic Circuit IFC Fluidigm Corporation catalogue number BMK M96 96 3 3 REQUIRED EQUIPMENT NOT SUPPLIED 4 4 4 Pipettes covering the range from 1 uL to 1000 uL Multi channel pipettes recommended range 1 10 uL 50 100 uL or 50 200 Vortex Centrifuge for plates Microcentrifuge for tubes Freezing block 20 C for enzyme handling Thermal cycler with gt Heated lid Temperature range from 50 C to 95 C Validated for O 1mL volumes important 96 well format recommended Olink has tested ABI 2720 and ABI Veriti 96 Well Thermal Cycler Refrigerator or cold room 2 C to 8 C Fluidigm BioMark or BioMark HD System Fluidigm IFC controller HX Proseek Multiplex 7 8 3 4 SOFTWARE FOR ANALYSIS Each Proseek Multiplex experiment will generate 9216 data points It is advisable to use a multivariate statistical software for data analysis We recommend the GenEx software developed by Mul
17. re 2 and select a location for each sample Day 1 INCUBATION 1 Thaw samples vortex and spin down the content at 150 x g 1 min at room temperature 2 Thaw the Incubation Stabilizer from the Proseek Multiplex Detection Kit 996 box vortex and spin briefly 3 Thaw the Interplate Control and Negative Control from the Proseek Multiplex Controls box vortex and spin briefly 4 Prepare the following ncubation mix in a microcentrifuge tube Vortex and spin each reagent before transfer to the mix Note Pipette the Incubation Solution carefully to avoid foaming Please note that Incubation mix per 96 well plate Teva me eva biben Incubation Solution 280 0 changed from previous Incubation Stabilizer 40 0 version v 1 3 A probes 40 0 B probes 40 0 Total 400 0 5 Vortex the ncubation mix briefly and spin down the content Transfer 47 uL per well of the ncubation mix by using reverse pipetting to an 8 well strip 6 Use a multi channel pipette to transfer 3 uL of the ncubation mix 7 Add 1 uL of each sample to the bottom of the well of the ncubation Plate according to your plate layout 8 Add 1 ul of Negative Control to the bottom of each well in position C12 D12 and E12 according to the plate layout in Figure 2 9 Add 1 uL of Interplate Control to the bottom of each well in position F12 G12 and H12 10 Seal the ncubation Plate with an adhesive plastic film It is important 11 from
18. tiD Analyses AB GenEx offers an easy to use Olink Wizard to guide you through the different steps of data preprocessing followed by a wide variety of statistical analysis such as hierarchical clustering methods principal components analysis ANOVA tests and more For more information please contact support olink com 3 5 DOWNLOADS The list of proteins can directly be imported into the Fluidigm Analysis software as a plt file Download the Detector Setup plt file corresponding to your panel at www olink com products proseek multiplex downloads data analysis files For data normalization without the Olink Wizard for GenEx please download the DataPreprocessing xlsx file corresponding to your panel at www olink com 3 6 TECHNICAL CONTROLS Each Proseek Multiplex 96x96 kit contains four internal controls Incubation Control 1 Incubation Control 2 Extension Control and Detection Control The internal controls are included in the assay reagents and hence added to each sample to be tested The two Incubation Controls are two control immunoassays measuring spiked in non human antigens These controls measure the variation in all three steps of the assay Incubation Extension and Detection The Extension Control is an antibody labeled with both pair of oligonucleotides needed for amplification The Extension Control is not dependent on the proximity of two different antibodies thus not affected during Incubation This control is used for
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