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QuantiGene® Plex 2.0 Reagent System

1. Repeat this procedure for all temperature settings used in the assay Verifying the Optimal Temperature To verify the optimal temperature Step Action 1 Perform a temperature titration of your assay to ensure the temperature setup is optimal We recommend performing a 2 C titration from the recommended assay temperature An example experiment is shown below QuantiGene Plex 2 0 Reagent System User Manual Page 37 Appendix IV Validating Alternative Shaking Incubators Page 38 To verify the optimal temperature continued Step Action 2 Analyze the results for the highest signal lowest background and lowest limit of detection Sample Volume uL 53 C 55 C 57 C 80 40 20 10 5 0 background Verifying Uniformity To verify the uniformity Step Action 1 Load the same amount of sample across the entire plate 2 Run the assay and compare the well to well variation The average CV across all wells should be less than 15 QuantiGene Plex 2 0 Reagent System User Manual Appendix V Automated Filter Plate Washing Method optional Appendix V Automated Filter Plate Washing Method optional About Automated Here we provide guidelines for setting up and running the BioTek ELx50 8FMW Washing Microplate Strip Washer IMPORTANT Use of this automated washing system requires additional pur
2. QuantiGene Plex 2 0 Reagent System User Manual Capturing Target RNA from Cultured Cell and Whole Blood Lysates To capture target RNA from cultured cell and whole blood lysates continued Step Action 5 Prepare an appropriate volume of Working Plex Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 overage Use the table below as a guide IMPORTANT Vortex at maximal speed Capture Beads for 30 seconds before addition Order of 1 Well 48 Wellsa 96 Wellsa Addition Reagent uL uL uL 1 RNase free water 5 2 349 697 2 Lysis Mixture 6 6 442 884 3 Blocking Reagent 2 134 268 4 Proteinase K 0 2 13 27 5 2 0 Capture Beads 1 67 134 6 2 0 Probe Set 5 335 670 Total 20 1 340 2 680 a Includes 40 overage Vortex Working Plex Set for 30 seconds to mix then dispense into the Hybridization Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 20 uL Working Plex Set into each well of the Hybridization Plate For 48 wells or more a Using a single channel pipette transfer Working Plex Set to a 25 mL capacity divided reagent reservoir Note Do not pour or reagent shortage will occur b Using a multichannel pipette and new tips for each transfer dispense 20 uL of Working Plex Set into each well of the Hybridization Plate IMP
3. We provide recommendations for typical starting sample inputs for each sample type see Capturing Target RNA on page 15 Using these recommendations as a guide perform a 2 fold dilution series of your sample and verify that the resulting assay signals are linearly proportional to sample input For more information see Determining Assay Linearity on page 12 Optimal QuantiGene Plex assay performance depends on the complete release and stabilization of the RNA from the cells and protein complexes Incomplete cell lysis may result in Poor assay precision High CV values Non linear assay If any of these conditions occur your samples may not be completely lysed Complete cell lysis depends on Correct ratio of cells to lysis solution Working Lysis Mixture or Working Homogenization Solution Method used to lyse the cells Guidelines on cell lysis solution ratios and lysis methods can be found in the QuantiGene Sample Processing Kit Product Inserts We strongly recommend running an optimization experiment in which different amounts of samples are used with a constant amount of appropriate lysis solution to ensure assay linearity and precision Determining the Correct Cell Lysis Solution Ratio For example if you are lysing cell cultures prepare lysate at 400 cells uL To optimize this make additional lysates at 200 and 800 cells uL Make serial dilutions of each lysate and evaluate for viscosity assay CV and ass
4. n a D ab a hn Q bal kO St 39 Procedure for Cell and RNA Sample Types 2 000020 2005s 40 Procedure for Tissue Blood and FFPE Sample Types 41 Appendix VI Blank Plate Maps KK KK KI KK K KK KI KI KI K K KK KIR KK KK 42 QuantiGene Plex 2 0 Reagent System User Manual Section Introduction Section I Introduction About This User Manual Who Should Read This manual is for anyone who has purchased QuantiGene Plex 2 0 Assay Kits from This Manual Panomics to perform the QuantiGene Plex assay for any of the following sample types Cultured cells Blood samples whole blood PAXgene blood dried blood spots Fresh frozen or formalin fixed paraffin embedded FFPE animal tissues Purified RNA What This Manual This manual provides recommendations and step by step procedures for the Covers following Experimental design and data analysis QuantiGene Plex 2 0 assay Troubleshooting Safety Warnings All chemicals should be considered potentially hazardous We recommend that this product and and Precautions its components be handled by those trained in laboratory techniques and used according to the principles of good laboratory practice For research use only Not for use in diagnosis of disease in humans or animals Contacting Panomics Technical Help For technical questions please contact our technical support group by telephone at 1 877 726 6642 option 3 or
5. ABgene heat sealer a Center the seal on the Hybridization Plate with white side facing up and silver side contacting the surface of the plate b Place the Hybridization Plate squarely onto the accessory plate carrier c Press down firmly to seal for 5 full seconds d Turn plate 180 degrees and repeat step c 9 Place an inverted Plate Lid into each of the two plate carriers in the Vortemp shaking Incubator then place the Hybridization Plate on top of the inverted Plate Lid If running a single plate place another filter plate with a plate seal in the second position for balance Incubate for 18 22 hours at 54 C 1 C and 600 rpm IMPORTANT Use a QuantiGene Incubator Temperature Validation Kit to verify the assay temperature In the case of Vortemp instruments with the inverted plate lids in place the Vortemp digital display and the actual temperature measured by the QuantiGene Incubator Temperature Validation Kit thermocouple inserted into the mock Hybridization Plate may differ by 4 C or more QuantiGene Plex 2 0 Reagent System User Manual Capturing Target RNA from Fresh Frozen or FFPE Animal Tissue Homogenates Capturing Target RNA from Fresh Frozen or FFPE Animal Tissue Homogenates Procedure To capture target RNA from tissue homogenates Step Action 1 Prepare the following reagents Probe Set and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect cont
6. KK KK KK eae ra Accessory Reagents 4 lk kk kk kk E kk eee eens 8 Required Reagents and Equipment kk kk KK KK KK KK KK e eee eee K KK K 8 About Required Materials nanana kk kK KK KK RR KK KK KK KIR eee 8 Equipment and Disposables kk kk kk kk KK KK KK KK KI KK KK KK KK KI KK eee 8 Guidelines for Assay Design and Analysis W kK kK KK KK KK KK eee 9 OVGOIVIOW 22 a ac des lee Reed b eee eed beers 9 Optimizing Sample Input lille 9 Determining Complete Cell Lysis lille 10 Replicate Recommendations kk kk kK KK KK KK KK KK KRI KK KK KK 10 Recommended Assay Controls kk kk kk KK kK KK KK KK KK KK KI KK KK KK KK KK kK k 11 Assay Background Control kk kk kk KK KK KK ees 11 Use of Housekeeping GeneS 00 kK kK KK KK KK KK KK KK KK KK KK k 11 Data Analysis Guidelines lll lille 11 Calculating Assay Precision es r terao awan eee 11 Calculating Assay Limit of Detection 0 0 0 0 cee eee 11 Determining Assay Linearity 0 0 00 KK KK KK KK KK KK eae 12 Normalizing Gene Expression Data KK KK KK KK ee eee 12 Calculating Fold Change 20 000 KK KK KK KK KK KK eh 12 Section Il QuantiGene Plex 2 0 Assay Procedure J LL LV kk kk YY kk RE kk kk kk Ek kK kK ka 13 About the Assay Procedure 1 2 2 kk kk kk KK E EK KK KI KK KK kk hne 13 Urn D 13 Instrument and Equipment Setup kk kk kk kk KK KK KK KK KK KI KK KK eee 14 Filtration System Se
7. Subtract the AVG assay background signal from the AVG signal of technical replicates for each RNA Calculate the ratio of background subtracted AVG RLU from sequential sample dilutions for each RNA Observed values should be within 20 of the expected ratio of 100 80 120 For example of Obs Exp Obs Exp x 100 where Obs Observed background subtract average RLU and Exp Expected fold change based on dilution 3 fold serial Signal dilution of background the celllysate subtracted Observed Expected pL MFI fold change fold change Obs Exp 60 3100 3 10 3 103 20 1000 2 70 3 90 6 6 370 Quantifiable signals are those signals within the assay linear range Normalizing Gene To normalize gene expression data Expression Data Step Action 1 Subtract the AVG assay background signal from the AVG signal of technical replicates for each RNA Divide the background subtracted AVG signals for each target RNA by the background subtracted AVG signal of the housekeeping RNA Note If multiple housekeeping RNAs are measured the geometric mean of background subtracted AVG housekeeping RNA signals may be used for data normalization Calculating To calculate fold change of an RNA target in treated versus untreated samples Fold Change Step Action 1 Normalize gene expression data as described above 2 Divide the normalized value for
8. beads before reading the plate Incorrect Luminex probe height Adjust the height of the probe following the procedures supplied with your Luminex system Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations Luminex or BioPlex system is clogged Refer to the troubleshooting guide provided with the system QuantiGene Plex 2 0 Reagent System User Manual Section III Troubleshooting Troubleshooting low bead count continued Probable Cause Recommended Actions Bubble introduction into Luminex fluidics Check Luminex probe for proper height then run instrument debubbling protocol Make sure every well contains 130 uL of SAPE Wash Buffer and verify the Luminex sample size is set to 100 uL Poor Assay Troubleshooting poor assay linearity Linearity Probable Cause Recommended Actions Inadequate sample preparation Refer to the appropriate sample processing kit product inserts for detailed procedures and troubleshooting Instrument saturation Signals 220 000 MFI on Luminex instruments are saturated Assay saturation When using 2 0 Capture Beads assay saturation occurs when RNA transcripts exceed approximately 5 000 000 When using 2 0 Mag Capture Beads assay saturation occurs when RNA transcripts exceed approximately 1 500 000 Perform serial dilution of sample to ensure appropriate fo
9. recommended input is gt 20 000 copies of each target RNA per well We recommend including 200 ng L yeast tRNA in IVT RNA dilutions to minimize RNA loss Page 22 QuantiGene Plex 2 0 Reagent System User Manual Capturing Target RNA from Total Purified or In Vitro Transcribed RNA To capture target RNA from purified RNA preparations continued Step Action 5 Prepare an appropriate volume of Working Plex Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 overage Use the table below as a guide IMPORTANT Vortex at maximum speed Capture Beads for 30 seconds before addition Order of 1 Well 48 Wellsa 96 Wells Addition Reagent uL uL uL 1 RNAse free water 38 7 2 593 5 186 2 Blocking Reagent 2 134 268 3 Lysis Mixture 33 3 2 231 4 462 4 2 0 Capture Beads 1 67 134 5 2 0 Probe Set 5 335 670 Total 80 5 360 10 720 a Includes 40 overage Vortex Working Plex Set for 30 seconds to mix then dispense into the Hybridization Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 80 uL Working Plex Set into each well of the Hybridization Plate For 48 wells or more a Using a single channel pipette transfer Working Plex Set to a 25 mL capacity divided reagent reservoir Note Do not pour or reagent shortage will occur b Using
10. replace QuantiGene 2 0 Wash Buffer with SAPE Wash Buffer 11 PRESS lt STOP gt TO QUIT QuantiGene Plex 2 0 Reagent System User Manual Page 41 Appendix VI Blank Plate Maps Appendix VI Blank Plate Maps Page 42 QuantiGene Plex 2 0 Reagent System User Manual
11. thaw at room temperature followed by incubation at 37 C for 30 minutes For tubes vortex briefly for plates pipette up and down 5 times then leave at room temperature until use IMPORTANT Do not put samples back on ice Transfer an appropriate volume 5 uL assay well plus 40 overage of Probe Set to a microfuge tube heat to 95 C for 5 minutes using a heat block then place on ice Pre warm Lysis Mixture at 37 C for 30 minutes followed by gentle swirling If appropriate based on the expression level of lowest abundance target or housekeeping RNA in the Plex Set dilute samples with Diluted Lysis Mixture 1 volume of Lysis Mixture plus 2 volumes nuclease free water prepared fresh so that the desired amount of sample is present in a volume of 80 uL assay well Use the table below as a guide and scale dilution volumes according to the number of assays to be run IMPORTANT Always verify that assay signals are within both the instrument and assay linear ranges For more information see Optimizing Sample Input on page 9 Determining Complete Cell Lysis on page 10 and Determining Assay Linearity on page 12 Recommended Sample Input Cultured Cells number of cells RNA copies per 2 0 Capture 2 0 Mag Capture Blood Lysate cell Beads Beads uL 1 32 000 20 000 80a 10 5 000 2 000 80 100 500 200 gt 1000 lt 50 lt 20 lt 0 08 a May not have the sensitivity required
12. the treated sample by the normalized value for the untreated sample Page 12 QuantiGene Plex 2 0 Reagent System User Manual Section ll QuantiGene Plex 2 0 Assay Procedure Section ll QuantiGene Plex 2 0 Assay Procedure About the Assay Procedure Overview A general outline of the assay procedure is as follows Sample Preparation Refer to the appropriate QuantiGene Sample Processing Kit Product Insert for sample type specific instructions for preparing cultured cell and whole blood lysates or tissue homogenates Follow standard laboratory methods for purification of RNA Use samples immediately in QuantiGene Plex 2 0 assays or store at 80 C until use Instrument and Equipment Setup Setup filtration system Setup Luminex instrument Validate Luminex instrument setup and operation Target RNA Capture Dilute samples if appropriate Prepare Working Plex Sets Dispense Working Plex Sets samples and controls into Hybridization Plates Hybridize samples overnight Signal Amplification and Detection of Target RNA Transfer samples to Filter Plate and filter away unbound material Sequentially hybridize the 2 0 Pre Amplifier 2 0 Amplifier Label Probe and SAPE Analyze samples using a Luminex based instrument QuantiGene Plex 2 0 Reagent System User Manual Page 13 Instrument and Equipment Setup Instrument and Equipment Setup Filtration System Refer to the manufacturer s recommended
13. with the QuantiGene Plex Assay Kit Item Source Adjustable single and multi channel precision Major Laboratory Supplier MLS pipettes for dispensing 1 20 uL 20 200 uL and 200 1 000 uL QuantiGene Plex 2 0 Reagent System User Manual Guidelines for Assay Design and Analysis Item Source Reagent reservoirs 25 mL capacity divided 25 mL capacity 100 mL capacity VistaLab Technologies P N 3054 1004 or equivalent P N 3054 1002 or equivalent Corning Costar P N CLS 4873 or equivalent Microcentrifuge Eppendorf 5415D or equivalent Microplate centrifuge that can achieve 240 xg Eppendorf 5804R and rotor A 2 DWP or equivalent Vortex mixer MLS RNase free water MLS Microcentrifuge tubes MLS Vacuum filtration system Millipore P N MAVMO960R and or WP6111560 ELx50 Microplate Strip Washer with filter plate option BioTek ELx50 8FMW Shaking incubator with microplate adaptor capable of maintaining constant temperatures of 50 C 1 C and 54 C 1 C speed of 600 rpm and 3 mm orbit LabNet VorTemp 56 P N S 2056 Q a Microplate shaker capable of maintaining a speed of 600 rpm 0 12 inch orbit Labline model 4625 or equivalent Heat block at 95 C MLS Luminex or Luminex based instrument MiraiBio Bio Rad or other Luminex instrument provider Thermo seal heat sealer for 10 plate Assay Kits ABGene P N AB 0384 and AB 05
14. your Plex Set so that a single sample input results in a linear response for all targets See Appendix Ill Common Housekeeping Genes and their Relative Expression Levels on page 36 for a list of common housekeeping genes and their relative abundance Signals for housekeeping genes can be used to normalize gene expression data across samples Include one or more housekeeping genes in the design of every Plex Set Data Analysis Guidelines Calculating Assay The Coefficient of Variation CV is a measure of assay precision QuantiGene Plex Precision 2 0 Assay CVs are typically less than 15 for technical replicates To determine the assay CV Step Action 1 Run technical replicates of each sample 2 Calculate the average signal AVG of technical replicates for each RNA 3 Calculate the standard deviation SD of signals from technical replicates for each RNA 4 Calculate the CV CV SD AVG 100 Calculating Assay Calculate assay limit of detection LOD for each RNA as follows Limit orpetecdon LOD AVG RLU of assay background control wells 3X SD of assay background signals Assay signals below LOD should not be used to draw quantitative conclusions about gene expression QuantiGene Plex 2 0 Reagent System User Manual Page 11 Data Analysis Guidelines Determining To determine the assay linearity Assay Linearity a ep Action 1 Run a dilution series of your sample 2
15. 2 Aspirate 12 sec A2 Tissue Blood and FFPE Sample Types First Wash 1 Link 05 wash overnight reaction Second Wash 1 Link 06 wash out unbound PreAmp and Amp Third Wash 1 Link 07 wash out unbound Label Probe Fourth Wash 1 Link 08 wash out unbound SAPE Aspirate 13 sec A4 Aspirate 17 sec A5 Aspirate 13 sec A4 Aspirate 13 sec A4 Dispense 200 uL Dispense 200 uL Dispense 200 uL Dispense 200 uL D1 D1 D1 D1 Aspirate 13 sec A4 Aspirate 13 sec A4 Aspirate 13 sec A4 Aspirate 13 sec A4 Dispense 200 uL Dispense 200 uL Dispense 200 uL Dispense 200 uL D1 D1 D1 D1 Aspirate 13 sec A4 Aspirate 20 sec A6 Aspirate 20 sec A6 Aspirate 20 sec A6 Dispense 200 uL Dispense 130 uL D1 D2 Aspirate 17 sec A5 To wash cell and RNA samples Step Action 1 Verify the washer is set at LOW vacuum level and leave the vent port on the carrier open Place QuantiGene 2 0 Wash Buffer in the appropriate bottle Run the Prime program New Buffer Prime Run Link program 01 First Wash overnight hybridization reaction Main Menu RUN MORE LINK LINK SELECT LINK 01 FIRST WASH ENTER NUMBER CF STRIP PLACE PLATE IN CARRIER AND PRESS lt START gt QuantiGene Plex 2 0 Reagent System User Manual Appendix V
16. 63 1000 or equivalent QuantiGene Incubator Temperature Validation Kit Panomics P N QS0517 a If an alternative is used see Appendix IV Validating Alternative Shaking Incubators on page 37 for guidelines on validating a non Vortemp shaking oven Guidelines for Assay Design and Analysis Overview Here we provide information and guidelines on the following Optimizing Sample Input Optimizing sample input Determining complete cell lysis Replicate recommendations Assay background controls Use of housekeeping genes 9 9 Data analysis The sensitivity of the QuantiGene Plex 2 0 assay is approximately 5 000 RNA copies per target per well when using 2 0 Capture Beads polystyrene and 1 500 RNA copies per target per well when using 2 0 Mag Capture Beads magnetic QuantiGene Plex 2 0 Reagent System User Manual Page 9 Guidelines for Assay Design and Analysis Determining Complete Cell Lysis Replicate Recommendations Page 10 When running a sample type for the first time and or using a new target specific Plex Set we strongly recommend that you optimize sample input to ensure the signals are within the assay and instrument linear range Luminex instruments exhibit saturation at approximately 20 000 MFI In our experience assay linear working ranges are approximately 5K 5 000K RNA transcripts when using 2 0 Capture Beads and 1 5K 1 500K RNA transcripts when using 2 0 Mag Capture Beads
17. Automated Filter Plate Washing Method optional Procedure for Tissue Blood and FFPE Sample Types To wash cell and RNA samples continued Step Action 5 Run Link program 02 Second Wash remove unbound Pre Amplifier 6 Run Link program 02 Second Wash remove unbound Amplifier 7 Run Link program 03 Third Wash remove unbound Label Probe 8 Place SAPE Wash Buffer in the appropriate bottle 9 Run the Prime program New Buffer Prime 10 Run Link Program 04 Fourth Wash replace QuantiGene 2 0 Wash Buffer with SAPE Wash Buffer 11 PRESS STOP TO QUIT To wash tissue blood and FFPE samples Step Action 1 Verify the washer is set at LOW vacuum level and leave the vent port on the carrier open Place QuantiGene 2 0 Wash Buffer in the appropriate bottle Run the Prime program New Buffer Prime Run Link program 05 First Wash 1 overnight hybridization reaction Main Menu RUN MORE LINK LINK SELECT LINK 05 FIRST WASH ENTER NUMBER OF STRIP gt PLACE PLATE IN CARRIER AND PRESS lt START gt Run Link program 06 Second Wash_1 remove unbound Pre Amplifier Run Link program 06 Second Wash_1 remove unbound Amplifier Run Link program 07 Third Wash_1 remove unbound Label Probe Place SAPE Wash Buffer in the appropriate bottle O NIO Oc Run the Prime program New_Buffer_Prime Run Link Program 08 Fourth Wash_1
18. ERE Qa0QRaGe6 If using components from a 10 plate Assay Kit seal the Hybridization Plate s with Heat Sealing Foil using the ABgene heat sealer a Center the seal on the Hybridization Plate with white side facing up and silver side contacting the surface of the plate b Place the Hybridization Plate squarely onto the accessory plate carrier c Press down firmly to seal for 5 full seconds d Turn plate 180 degrees and repeat step c 9 Place an inverted Plate Lid into each of the two plate carriers in the Vortemp shaking Incubator then place the Hybridization Plate on top of the inverted Plate Lid If running a single plate place another filter plate with a plate seal in the second position for balance Incubate for 18 22 hours at 54 C 1 C and 600 rpm IMPORTANT Use a QuantiGene Incubator Temperature Validation Kit to verify the assay temperature In the case of Vortemp instruments with the inverted plate lids in place the Vortemp digital display and the actual temperature measured by the QuantiGene Incubator Temperature Validation Kit thermocouple inserted into the mock Hybridization Plate may differ by 4 C or more QuantiGene Plex 2 0 Reagent System User Manual Signal Amplification and Detection of RNA Targets Signal Amplification and Detection of RNA Targets About this These instructions are for processing one 96 well plate using multichannel pipettes Procedure and reagent reservoirs To process fe
19. FRC Low ATP6V1A GUSB 8 000 80 000 2 000 80 000 HMBS TBP TNX2 Page 36 QuantiGene Plex 2 0 Reagent System User Manual Appendix IV Validating Alternative Shaking Incubators Appendix IV Validating Alternative Shaking Incubators About Alternative Alternative equipment must meet the minimum specifications stated in Required Incubators Reagents and Equipment on page 8 Systems Tested by Panomics We have validated 2 systems using this procedure Both show 2 fold lower sensitivity than the Vortemp incubator WVRFM1350 incubator oven and Labnet orbit P4 microplate shaker ATRBiotech Multitron Il Micro shaker incubator www artbiotech com Validating Before You Start Procedure This procedure requires 3 Panomics thermometers QuantiGene Incubator Temperature Validation Kit P N QS0517 Calibrating the Oven To calibrate the oven Step Action 1 Place 3 Panomics thermometers into the oven spreading them evenly throughout the plate platform Set the oven to the recommended assay temperature After 4 hours record the readings of the 3 thermometers Adjust the oven temperature raise the temperature if readings are lower lower the temperature if the readings are higher Wait another 2 hours and read the thermometers Adjust if necessary Repeat steps 4 5 until the oven reaches the recommended assay temperature Note the oven setting NIO ao
20. OCOL PROTOCOL ADVANCE ADVANCE WINDOW WINDOW SETTING enter SETTING enter DD gate values DD gate values Bio Plex Standard Automatically Standard Automatically 100 45 100 5 0 BioRad SELECT set SELECT set Page 34 QuantiGene Plex 2 0 Reagent System User Manual Appendix Il Recommended Maintenance for Luminex Instruments 2 0 Capture Bead polystyrene 2 0 Mag Capture Bead magnetic Software Luminex xMAP Luminex MagPlex Instrument Bead Platform and Bead Sample Size Timeout Events Bead Supplier Calibration DD gate Bead Calibration DD gate uL sec region Applied Standard 4 300 10 000 Standard 5 000 25 000 100 40 100 Station Applied TEMPLATE TEMPLATE Cytometry SET UP gt enter SET UP gt enter DD DD gate value gate value a You must use MagPex calibrator beads for calibration MagPlex Calibration Beads Luminex P N MCAL1 05 and MagPlex Control Beads P N MCON1 05 Appendix Il Recommended Maintenance for Luminex Instruments About Luminex Maintenance For optimal results we strongly recommend that you clean the sample probe needle on a regular basis according to the manufacturer s recommendations Probe needles After Each Run that have rusty or salt deposits should be replaced Luminex P N CN 0007 01 Clean the probe needle after each run as indicated in the following table Step Number of Times Solution Sanitize 2x 20 bleach Backflush 3
21. ORTANT Include 3 wells for assay background control Using a new pipette tip for each transfer add 80 uL cell lysate or diluted cell lysate to each well of the Hybridization Plate containing Working Plex Set IMPORTANT Add 80 uL of Diluted Lysis Mixture 1 volume of Lysis Mixture plus 2 volumes of nuclease free water to 3 wells for assay background control QuantiGene Plex 2 0 Reagent System User Manual Page 17 Capturing Target RNA from Cultured Cell and Whole Blood Lysates Page 18 To capture target RNA from cultured cell and whole blood lysates continued Step Action 8 Use one of the following methods to seal the plate If using components from a 1 or 3 plate Assay Kit a Tightly seal the Hybridization Plate using the Sealing Mat IMPORTANT Ensure that each individual well of the sealing mat has been snapped into place Failure to completely seal the plate may result in excessive evaporation during the overnight incubation b Cover the plate with the plate lid and secure it with a clamp at each end as shown below Note If clamped plates do not fit in the incubator holder heavy duty rubber bands may be substituted for the clamps A Plate Lid 2 Sealing Mat Clamp 1 Clamp UU ud s Overnight Hybridization Plate B Side View Top View EERE Qa0QRaGe6 If using components from a 10 plate Assay Kit seal the Hybridization Plate s with Heat Sealing Foil using the
22. Plate into the instrument and read the 2 wells View the window with the bead regions and DD gate The expected results are Signals for the expected beads show up on the bead map Average bead count is greater than 50 region Single peak in the DD gate window with signals within the set DD gate region Capturing Target RNA About Capturing This section provides three protocols for capturing target RNA based on sample type Target RNA a Cultured cell and whole blood lysates b Fresh frozen or FFPE animal tissue homogenates C Total RNA mRNA or in vitro transcribed RNA preparations Refer to the appropriate protocol for your sample type QuantiGene Plex 2 0 Reagent System User Manual Page 15 Capturing Target RNA from Cultured Cell and Whole Blood Lysates Capturing Target RNA from Cultured Cell and Whole Blood Lysates Page 16 Procedure To capture target RNA from cultured cell and whole blood lysates Step Action 1 Prepare the following reagents Probe Set and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents at the bottom of the tube Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minutes followed by gentle swirling Capture Beads Take out of storage right before use and protect from light Proteinase K Take out of storage right before use and place on ice Cultured cell or whole blood lysates If frozen
23. QuantiGene Plex 2 0 Reagent System User Manual Panomics Panomics Inc QuantiGene Plex 2 0 Reagent System User Manual Copyright Copyright 2008 Panomics Inc All rights reserved Trademarks QuantiGene is a registered trademark exclusively licensed to Panomics Inc All other trademarks belong to their respective owners Citing QuantiGene Plex Reagent System in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene Plex 2 0 Reagent System If a paper cites a QuantiGene Plex 2 0 Reagent System product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information U S Corporate Headquarters Panomics Inc 6519 Dumbarton Circle Fremont CA 94555 Toll Free 877 PANOMICS 1 877 726 6642 Direct 1 510 818 2600 Fax 1 510 818 2610 Email info panomics com Email order
24. Reagent to each assay well c Very gently seal the Filter Plate with a foil Plate Seal then blot the bottom of the Filter Plate on a clean dry paper towel d Place the Filter Plate on the inverted Plate Lid and incubate for 1 hour in the Vortemp shaking incubator at 50 C 1 C and 600 rpm QuantiGene Plex 2 0 Reagent System User Manual Page 27 Signal Amplification and Detection of RNA Targets Hybridizing the To hybridize the 2 0 Amplifier 2 0 Amplifier Step Action 1 Prepare 2 0 Amplifier Working Reagent a Centrifuge the 2 0 Amplifier briefly to collect the contents at the bottom of the tube b Add 36 uL of 2 0 Amplifier to 12 mL of Amplifier Diluent c Invert several times to mix IMPORTANT The Amplifier Diluent is a viscous solution Pipet carefully to ensure that the entire contents are expelled from the pipette and that the working reagent is completely transferred to the reagent reservoir 2 Wash away the unbound 2 0 Pre Amplifier a Remove the Filter Plate from the shaking incubator b Remove the foil Plate Seal from the assay wells and filter completely then turn off vacuum c Carefully add 200 uL of 2 0 Wash Buffer to each of the wells filter completely then turn vacuum off Repeat step 2c for a total of two washes Blotthe top and bottom of the Filter Plate to completely remove all traces of 2 0 Wash Buffer and return to the Filter Plate Holder f Immediately proceed to the
25. a multichannel pipette and new tips for each transfer dispense 80 uL of Working Plex Set into each well of the Hybridization Plate IMPORTANT Include 3 wells for assay background control Using a new pipette tip for each transfer add 20 uL RNA sample to each well of the Hybridization Plate containing the Working Plex Set IMPORTANT Add 20 uL RNase free water to 3 wells for assay background control For IVT RNA background controls add 20 uL RNase free water containing 200 ng uL yeast tRNA QuantiGene Plex 2 0 Reagent System User Manual Page 23 Capturing Target RNA from Total Purified or In Vitro Transcribed RNA Page 24 To capture target RNA from purified RNA preparations continued Step Action 8 Use one of the following methods to seal the plate If using components from a 1 or 3 plate Assay Kit a Tightly seal the Hybridization Plate using the Sealing Mat IMPORTANT Ensure that each individual well of the sealing mat has been snapped into place Failure to completely seal the plate may result in excessive evaporation during the overnight incubation b Cover the plate with the plate lid and secure it with a clamp at each end as shown below Note If clamped plates do not fit in the incubator holder heavy duty rubber bands may be substituted for the clamps A Plate Lid 2 Sealing Mat Clamp 1 Clamp UU ud s Overnight Hybridization Plate B Side View Top View E
26. ate Time 13 sec 17 sec 20 sec QuantiGene Plex 2 0 Reagent System User Manual Page 39 Appendix V Automated Filter Plate Washing Method optional Procedure for Cell and RNA Sample Page 40 Types To create a program for QuantiGene 2 0 assay in a 96 well Filter Plate continued Step Action 4 Create Link programs for day 2 processing Main Menu gt DEFINE gt CREATE gt MORE gt LINK gt Enter NAME 1st WASH gt Select LINK 01 gt ENTER ASPIR A1 gt ENTER DISP D1 gt ENTER ASPIR A1 gt ENTER DISP D1 gt ENTER ASPIR A1 gt ENTER DISP D1 gt ENTER ASPIR A2 Cell Lysates and Total RNA Sample Types First Wash Link 01 wash overnight reaction Second Wash Link 02 wash out unbound PreAmp and Amp Third Wash Link 03 wash out unbound Label Probe Fourth Wash Link 04 wash out unbound SAPE Aspirate 8 sec A1 Aspirate 12 sec A2 Aspirate 8 sec A1 Aspirate 8 sec A1 Dispense 200 uL D1 Dispense 200 uL D1 Dispense 200 uL D1 Dispense 200 uL D1 Aspirate 8 sec A1 Aspirate 8 sec A1 Aspirate 8 sec A1 Aspirate 8 sec A1 Dispense 200 uL D1 Dispense 200 uL D1 Dispense 200 uL D1 Dispense 200 uL D1 Aspirate 8 sec A1 Aspirate 15 sec A3 Aspirate 15 sec A3 Aspirate 15 sec A3 Dispense 200 uL D1 Dispense 130 uL D
27. ay linearity Optimizing the Lysis Method Start with the recommended procedure to get optimal results Subsequently you can modify the procedure as needed to simplify the process Regardless of the process used evaluate the assay linearity and precision to ensure optimal and consistent results Technical replicates are replicate assays from a single sample For example a cell lysate that is divided into several portions and each portion run in the same QuantiGene Plex 2 0 assay Biological replicates are replicate assays from biologically equivalent samples For example cells grown in different wells that are subjected to the same treatment lysed independently then run as distinct samples in the QuantiGene Plex 2 0 assay QuantiGene Plex 2 0 Reagent System User Manual Recommended Assay Controls We recommend running 3 technical replicates of each distinct biological sample Recommended Assay Controls Assay Assay background is the signal median fluorescence intensity MFI generated by the Background assay components in the absence input RNA Control Run an assay background control in triplicate for each Plex Set used in each experiment Use of A Housekeeping gene is a target gene that is stably expressed under all experimental Housekeeping conditions evaluated An important aspect of a housekeeping gene is its relative Genes abundance Ideally housekeeping genes expression levels should be similar to the target genes in
28. chase of QuantiGene Plex 2 0 Wash Buffer and SAPE Wash Buffer Each use requires a 40 mL priming volume IMPORTANT Review the BioTek ELx50 General Usage Guidelines before setting up and running the QuantiGene assay Creating a To create a program for QuantiGene 2 0 assay in a 96 well Filter Plate Program Step Action 1 Create Prime Program called New Buffer Prime and enter the following settings Prime Volume 40 mL Prime Flow Rate 6 600 uL well sec Soak after Prime No 2 Create individual Dispense Programs D1 and D2 Main menu DEFINE gt CREATE gt DISP gt Select DISP program gt Enter program name D1 Parameter D1 D2 Dispense Volume 200 uL 130 uL Dispense Flow Rate 5 500 uL well sec 5 500 uL well sec Dispense Height 120 15 240 mm 120 15 240 mm Horizontal Dispense Position 0 0 mm 0 0 mm Prime Before Start No No 3 Create 6 individual Aspiration Programs A1 A3 for cells and RNA and A4 A6 for tissue blood and FFPE Main Menu gt DEFINE gt Create gt ASPIR gt Select ASPIR program gt Enter program name A1 Cell Lysates and Total RNA Sample Types Parameter A1 A2 A3 Aspirate Type Bottom BOT Bottom BOT Bottom BOT Aspirate Time 8 sec 12 sec 15 sec Tissue Blood and FFPE Sample Types Parameter A4 A5 A6 Aspirate Type Bottom BOT Bottom BOT Bottom BOT Aspir
29. combines a flow cytometer fluorescent dyed microspheres beads lasers and digital signal processing to allow multiplexing of up to 100 unique assays within a single sample The QuantiGene Plex assay is compatible with all Luminex based instruments currently available Assay Principal The QuantiGene Plex 2 0 system utilizes fluorescent microspheres Capture Beads as a support to capture specific RNA molecules The ability to quantify multiple target specific RNA molecules in a single sample lies in the design of the Probe Sets For each RNA molecule of interest an oligonucleotide Probe Set containing three types of synthetic probes Capture Extenders CEs Label Extenders LEs and Blockers BLs that hybridize and span contiguous sequences of the target RNA is provided The CEs discriminate among the different Capture Beads within the bead array while capturing via cooperative hybridization the target RNA Signal amplification is mediated by DNA amplification molecules that hybridize to the tails of the LEs Each amplification unit contains multiple hybridization sites for biotinylated Label Probes that bind Streptavidin conjugated R Phycoerythrin SAPE The resulting fluorescence signal associated with individual Capture Beads is read on a Luminex flow cytometer Signal is reported as median fluorescence intensity MFI and is proportional to the number of target RNA molecules present in the sample QuantiGene Plex 2 0 Reagent System Use
30. copies per cell Tissue Homogenates pL 1 40a 10 40 100 gt 1 000 lt 0 4 a May not have sensitivity required QuantiGene Plex 2 0 Reagent System User Manual Page 19 Capturing Target RNA from Fresh Frozen or FFPE Animal Tissue Homogenates To capture target RNA from tissue homogenates continued Step Action 5 Prepare an appropriate volume of Working Plex Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 overage IMPORTANT Vortex at maximal speed Capture Beads for 30 seconds before addition Order of 1 Well 48 Wellsa 96 Wellsa Addition Reagent pL pL pL 1 RNase free water 18 5 1 240 2 479 2 Lysis Mixture 33 3 2 231 4 462 3 Blocking Reagent 2 134 268 4 Proteinase K 0 2 13 27 5 2 0 Capture Beads 67 134 6 2 0 Probe Set 5 335 670 Total 60 4 020 8 040 a Includes 4096 overage Vortex Working Plex Set for 30 seconds to mix then dispense into the Hybridization Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 60 uL Working Plex Set into each well of the Hybridization Plate For 48 wells or more a Using a single channel pipette transfer Working Plex Set to a 25 mL capacity divided reagent reservoir Note Do not pour or reagent shortage will occur b Using a multichannel pipette and new
31. dividual components supplied Kit components have a shelf life of 6 months from the date of receipt Each Assay Kit contains the following components Component Description Storage Proteinase Ka Proteinase K in aqueous buffered solution 20 C Blocking Reagent Aqueous buffered solution containing a 20 C preservative Label Probe Biotinylated oligonucleotide in aqueous 20 C buffered solution 2 0 Pre Amplifier DNA in aqueous buffered solution 20 C 2 0 Amplifier DNA in aqueous buffered solution 20 C Amplifier Diluent Aqueous buffered solution containing protein 2 8 C and a preservative Label Probe Diluent Aqueous buffered solution containing protein 2 8 C and a preservative SAPE Streptavidin conjugated R Phycoerythrin 2 8 C SAPE Diluent Aqueous buffered solution containing protein 2 8 C and a preservative Lysis Mixture Aqueous buffered solution containing a 15 30 C preservative 2 0 Wash Buffer Component 1 Aqueous solution 15 30 C QuantiGene Plex 2 0 Reagent System User Manual Page 7 Required Reagents and Equipment Accessory Reagents Each Assay Kit contains the following components continued Component Description Storage 2 0 Wash Buffer Component 2 Aqueous buffered solution 15 30 C SAPE Wash Buffer Aqueous buffered solution 15 30 C Filter Plate 96 well filter plate 15 30 C Filter Plate Holder 96 well clear bottom polyst
32. e made during the processing of the first sample IMPORTANT Use SAPE Wash Buffer The bead position on the DD gate might change if the incorrect buffer is used to suspend the Capture Beads during reading 2 0 Capture Bead polystyrene 2 0 Mag Capture Bead magnetic ANALYTE gt EDIT PANEL select xMAP ANALYTE gt EDIT PANEL select MagFlex Software Luminex xMAP Luminex MagPlex Instrument Bead Platform and Bead Sample Size Timeout Events Bead Supplier Calibration DD gate Bead Calibration DD gate uL sec region xPonent Luminex Standard Automatically Magnetic Automatically 100 40 100 v3 0 PROTOCOL set PROTOCOL set BEAD TYPE BEAD TYPE select Standard select magnetic IS100 Luminex Standard 4 300 15 000 Standard 5 000 25 000 100 40 100 v2 3 NEW BATCH NEW BATCH CREATE ASSAY CREATE ASSAY TEMPLATE gt Acq TEMPLATE gt Acq Detail enter DD Detail enter DD gate value gate value MasterPlex Standard 4 300 10 000 Standard 5 000 25 000 100 40 100 CT MiraiBio v1 0 ACQUISITION ACQUISITION SET UP gt SET UP SET UP gt SET UP enter DD gate enter DD gate value value MasterPlex Standard Automatically Magnetic Automatically 100 40 100 CT MiraiBio v1 2 ACQUISITION set ACQUISITION set TEMPLATE TEMPLATE SET UP gt BEAD SET UP gt BEAD TYPE select TYPE select Standard Magnetic Bio Plex Standard 4 300 10 000 Standard 5 000 25 000 100 45 100 4 0 BioRad RUN RUN PROT
33. e Diluent and mix thoroughly Expired reagents were used Reagents are good for 6 months from date of receipt Sub optimal assay conditions Follow the recommended incubation times and temperature Shake the Filter Plate during all incubations Verify the temperature of incubations throughout the procedure If you are not using the recommended Vortemp shaker see Appendix IV Validating Alternative Shaking Incubators on page 37 for validating alternative shakers Photobleaching of SAPE Protect SAPE from light throughout the procedure Incorrect wash buffer was used Use SAPE Wash Buffer to wash away unbound SAPE Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations Incomplete cell lysis Refer to the appropriate sample processing kit product inserts for detailed procedures and troubleshooting Significant RNA degradation Refer to the appropriate sample processing kit product inserts for detailed procedures and troubleshooting High Background Troubleshooting high background signal Signal Probable Cause Recommended Actions Probe Set was not heated prior to adding to the assay wells Heat the Probe Set to 95 C for 5 minutes then place on ice until use Sub optimal assay conditions Follow the recommended incubation times and temperature Shake the Filter Plate during all incubations Verify th
34. e temperature of incubations throughout the procedure If you are not using the recommended Vortemp shaker see Appendix IV Validating Alternative Shaking Incubators on page 37 for validating alternative shakers Hybridization mixture was left at room temperature for too long prior to overnight hybridization Minimize the setup time and ensure that the hybridization mixture sample is not left at room temperature for more than 10 minutes Expired reagents were used Reagents are good for 6 months from the date of receipt QuantiGene Plex 2 0 Reagent System User Manual Page 31 Section Ill Troubleshooting Low Assay Troubleshooting low assay precision Precision High CV Probable Cause Recommended Actions Inaccurate pipetting Use only calibrated precision pipettes Affix tips securely Use a new tip for each transfer Pipet slowly and carefully avoiding bubbles Residual Wash Buffer Filter Wash Buffer completely and blot the top and bottom of the Filter Plate with absorbent paper towels to remove residual Wash Buffer after the last wash in each series Non homogenous samples Warm samples to 37 C to dissolve any precipitate and vortex briefly before use If samples contain particulates centrifuge at high speed for 15 minutes then transfer supernatants to a new tube and repeat centrifugation and transfer step before use Incomplete cell lysis Refer to the app
35. email at techsupport panomics com US and Canada In Europe contact techsupport_europe panomics com In Asia Pacific contact techsupport asia panomics com For an updated list of FAQs and product support literature visit our website www panomics com QuantiGene Plex 2 0 Reagent System User Manual Page 5 About the QuantiGene Plex 2 0 Reagent System About the QuantiGene Plex 2 0 Reagent System Introduction QuantiGene Plex 2 0 Assay Basics Page 6 The QuantiGene Plex 2 0 Reagent System is designed to quantitate in a single well multiple target specific RNA molecules present in Cultured cell lysates Whole blood PAXgene blood RNA or dried blood spot lysates Fresh or frozen animal tissue homogenates FFPE animal tissue homogenates 9 9 9 9 Total RNA mRNA or in vitro transcribed RNA preparations Please refer to Panomics QuantiGene Sample Processing Kit Product Inserts for instructions on preparing cultured cell or whole blood lysates or animal tissue homogenates To prepare RNA follow standard laboratory methods Assay Technology The QuantiGene Plex 2 0 assay combines branched DNA bDNA signal amplification and multi analyte profiling beads xMAP technologies to enable the detection and quantitation of multiple mRNA targets simultaneously The bDNA assay is a hybridization based method of target specific RNA quantitation that amplifies signal rather than target RNA using labeled DNA probes The xMAP system
36. ents at the bottom of the tube Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minutes followed by gentle swirling Capture Beads Take out of storage right before use and protect from light Proteinase K Take out of storage right before use and place on ice Tissue homogenates If frozen thaw at room temperature followed by incubation at 37 C for 30 minutes For tubes vortex briefly for plates pipet up and down 5 times then keep at room temperature until use IMPORTANT Do not put samples back on ice Transfer an appropriate volume 5 uL assay well plus 40 overage of Probe Set to a microfuge tube heat to 95 C for 5 minutes using a heat block then place on ice Pre warm Lysis Mixture at 37 C for 30 minutes followed by gentle swirling If appropriate dilute tissue homogenates with Homogenizing Solution so that the desired amount of sample is present in a volume of 40 uL assay well based on the expression level of lowest abundance target or housekeeping RNA in the Plex Set Use the table below as a guide and scale dilution volumes according to the number of assays to be run IMPORTANT Always verify that assay signals are within both the instrument and assay linear ranges For more information see Optimizing Sample Input on page 9 Determining Complete Cell Lysis on page 10 and Determining Assay Linearity on page 12 Recommended Sample Input RNA
37. kk kk kk kk kk kk kk ak kk kk kk kk kk kk kk kk 31 Low Assay Signal or Poor Sensitivity uk KK KK RR eee 31 High Background Signal ercsi 2k huya n ka d wa n Zana ene 31 Low Assay Precision High CV sas sa k wada dreta W eae 32 Low Bead Count gt 3 ml hay ae ks ya kana KN ses 32 Poor Assay Linearity ka K lk a a n e 33 Clogged Filter Plate Wells 0 0 kk kk kK KRI KK KIR KIRR KI KK KK KK 33 Section AVA Appendices x 5 aw Aa ane k Ga nia eae ein xaneya Rec Win ka Pa a alla ye xa ata a V ya 34 Appendix Setting Up Luminex Instrument for QuantiGene Plex 2 0 Assays 34 Instrument Settings 4 44 sy wac kh bA a bak k belal eee ee eee 34 Appendix Il Recommended Maintenance for Luminex Instruments 35 About Luminex Maintenance 00 000 KK KK KK KI KI eee 35 After Each R n x serre xen etes fata dass E o ERE aye 35 Before Shut Down kk kk kk kk kK kk kK KK KK KK KK tees 35 Appendix III Common Housekeeping Genes and their Relative Expression Levels 36 Appendix IV Validating Alternative Shaking Incubators 2000200e 37 About Alternative Incubators 00 000 KK KK KK KK KK eee 37 Validating Procedure 00 aE O E eee 20l3 2 oU 37 Appendix V Automated Filter Plate Washing Method optional 39 About Automated Washing kk kk kk kk KK KK KI KK ren 39 Ciealihg Program gt Lese Wa k
38. l Probe a Transfer the Label Probe Working Reagent to a 25 mL capacity reagent reservoir Add 100 uL of the Label Probe Working Reagent to each assay well c Very gently reseal the Filter Plate with a foil Plate Seal then blot the bottom of the Filter Plate on a clean dry paper towel d Place the Filter Plate on the inverted Plate Lid and incubate for 1 hour in the shaking incubator at 50 C 1 C and 600 rpm Binding the SAPE To bind the SAPE Step Action 1 Prepare the SAPE Working Reagent a Briefly vortex SAPE to mix then briefly centrifuge to collect the contents at the bottom of the tube b Add 36 uL of SAPE to 12 mL of SAPE Diluent c Vortex for 15 seconds to mix and protect from light Wash away unbound Label Probe a Remove the Filter Plate from the shaking incubator b Remove the foil Plate Seal from the assay wells filter completely then turn off the vacuum c Carefully add 200 uL of 2 0 Wash Buffer to each assay well filter completely then turn off the vacuum d Repeat step 2c for a total of two washes blot the top and bottom of the Filter Plate and return to the Filter Plate Holder e Immediately proceed to the next step Bind the SAPE a Transfer the SAPE Working Reagent to a 25 mL capacity reagent reservoir b Add 100 uL of the SAPE Working Reagent to each assay well c Very gently reseal the Filter Plate with a foil Plate Seal then blot the bottom
39. late wells during manipulations or the contents of the wells might be forced through the filter resulting in reagent loss Minimize the exposure of beads to room light QuantiGene Plex 2 0 Reagent System User Manual Page 25 Signal Amplification and Detection of RNA Targets Hybridizing the 2 0 To hybridize the 2 0 Pre Amplifier Pre Amplifier Page 26 Step Action 1 Prepare 2 0 Pre Amplifier Working Reagent a Centrifuge the 2 0 Pre Amplifier briefly to collect the contents at the bottom of the tube b Add 36 uL of 2 0 Pre Amplifier to 12 mL of Amplifier Diluent c Invert several times to mix IMPORTANT The Amplifier Diluent is a viscous solution Pipet carefully to ensure that the entire contents are expelled from the pipette and that the working reagent is completely transferred to the reagent reservoir Pre wet the Filter Plate a Fill a 100 mL capacity reagent reservoir with 2 0 Wash Buffer b Seal unused wells with a foil seal c Place the Filter Plate on the Filter Plate Holder d Add 100 uL of 2 0 Wash Buffer to each well of the Filter Plate and incubate at room temperature for 1 minute e Transfer the Filter Plate to the vacuum manifold and filter the 2 0 Wash Buffer IMPORTANT Ensure vacuum pressure does not exceed 1 2 inches of Hg and filtration rate is 5 15 seconds Adjust pressure if necessary f Turn off vacuum and return the Filter Plate to the Filter Plate Holder Note See A
40. ld change is observed See Determining Assay Linearity on page 12 for more information Clogged Filter Troubleshooting clogged filter plate wells Plate Wells QuantiGene Plex 2 0 Reagent System User Manual Probable Cause Recommended Actions Inadequate sample preparation Refer to the appropriate sample processing kit product inserts for detailed procedures and troubleshooting Page 33 Section IV Appendices Section IV Appendices Appendix I Setting Up Luminex Instrument for QuantiGene Plex 2 0 Assays Instrument Calibrate the instrument using the standard PMT setting may be referred to as low by Settings some suppliers The key information in this table is related to the bead calibration required and setting of the DD gate based on the type Standard or Magnetic of Capture Beads being used Because of continual software updates we recommend that you contact your Luminex instrument supplier and verify the latest recommendations for setup of Standard and Magnetic xMAP Luminex beads When beads are injected into the flow cell a small percentage can clump and go through the flow cell as doublets The DD gate of Doublet Discriminator gate allows for discrimination of doublet formation When initially setting the DD gate follow the recommendations in the table below In some cases you might need to adjust the DD gate around the largest peak which represents the singlet beads Adjustments can b
41. mperature in the dark for up to 2 hours or at 4 C for 24 hours without shaking Proceed to the next step when you are ready to read the plate d Place on a shaking platform at room temperature and 600 rpm for 2 5 minutes then read immediately IMPORTANT If running more than 1 plate at a time leave the 2nd plate at room temperature in the dark without shaking Once the 1st plate has been read and the instrument wash protocol has been completed place the 2nd plate on a shaker platform at room temperature shaking at 600 rpm for 2 5 minutes then read immediately IMPORTANT Refer to Luminex Setup and Operation on page 14 for instrument setup and operation recommendations Page 30 QuantiGene Plex 2 0 Reagent System User Manual Section III Troubleshooting Section Ill Troubleshooting Low Assay Signal Troubleshooting low assay signal or poor sensitivity or Poor Sensitivity Probable Cause Recommended Actions Number of RNA transcripts below of detection limit Increase the sample input If using 2 0 Capture Beads polystyrene try using the high sensitivity setting for Luminex instruments This may result in a little more sensitivity at the low end but will impact the high end signals as instrument saturation occurs at 20 000 MFI Signal amplification reagent is incorrectly prepared Carefully add the correct amounts of 2 0 Pre Amplifier 2 0 Amplifier Label Probe and SAPE to the appropriat
42. next step 3 Start the 2 0 Amplifier hybridization a Transfer 2 0 Amplifier Working Reagent to a 25 mL capacity reagent reservoir b Add 100 uL of 2 0 Amplifier Working Reagent to each assay well c Very gently seal the Filter Plate with a foil Plate Seal then blot the bottom of the Filter Plate on a clean dry paper towel d Place the Filter Plate on the inverted Plate Lid and incubate for 1 hour in the Vortemp shaking incubator at 50 C 1 C and 600 rpm Hybridizing the To hybridize the Label Probe Label Probe Page 28 Step Action 1 Prepare the Label Probe Working Reagent a Centrifuge Label Probe briefly to collect the contents at the bottom of the tube b Add 36 uL of Label Probe to 12 mL of Label Probe Diluent c Vortex for 15 seconds to mix 2 Wash away the unbound 2 0 Amplifier a Remove the Filter Plate from the shaking incubator b Remove the foil Plate Seal from the assay wells and filter completely c Carefully add 200 uL of 2 0 Wash Buffer to each assay well filter completely then turn off the vacuum d Repeatstep 2c for a total of two washes blot the top and bottom of the Filter Plate and return to the Filter Plate Holder e Immediately proceed to the next step QuantiGene Plex 2 0 Reagent System User Manual Signal Amplification and Detection of RNA Targets To hybridize the Label Probe continued Step Action 3 Hybridize the Labe
43. ng The sensitivity setting is performed during calibration Refer to the table in Appendix I Setting Up Luminex Instrument for QuantiGene Plex 2 0 Assays on page 34 for information on setting up your Luminex instrument IMPORTANT Refer to the QuaniGene Plex 2 0 Plex Set Product Insert for the target bead region associations for your panel Page 14 QuantiGene Plex 2 0 Reagent System User Manual Capturing Target RNA Validating IMPORTANT Perform this procedure before you run the assay to ensure the Luminex Luminex instrument is setup correctly for your QuantiGene Plex assay Instrument Calibration and o verify the instrument calibration and setup Setup Step Action 1 Note Setup the Luminex instrument according to the guidelines in Appendix I Setting Up Luminex Instrument for QuantiGene Plex 2 0 Assays on page 34 Define a protocol with the appropriate bead regions and set to read 2 wells IMPORTANT Refer to the QuantiGene Plate 2 0 Plex Set Product Insert for the target bead associations for your panel Pre wet the Filter Plate by adding 100 uL SAPE Wash Buffer to 2 wells then filter Vortex 2 0 Capture Beads or 2 0 Mag Capture Beads at maximum speed for 30 seconds Add 1 uL of 2 0 Capture Beads or 2 0 Mag Capture Beads to 300 uL of SAPE Wash Buffer Vortex to mix Add 130 uL of the Capture Bead mixture into each of the 2 pre wet wells in the Filter Plate Insert the Filter
44. ng Foil using the ABgene heat sealer a Center the seal on the Hybridization Plate with white side facing up and silver side contacting the surface of the plate b Place the Hybridization Plate squarely onto the accessory plate carrier c Press down firmly to seal for 5 full seconds d Turn plate 180 degrees and repeat step c Place an inverted Plate Lid into each of the two plate carriers in the Vortemp shaking Incubator then place the Hybridization Plate on top of the inverted Plate Lid If running a single plate place another filter plate with a plate seal in the second position for balance Incubate for 18 22 hours at 54 C 1 C and 600 rpm IMPORTANT Use a QuantiGene Incubator Temperature Validation Kit to verify the assay temperature In the case of Vortemp instruments with the inverted plate lids in place the Vortemp digital display and the actual temperature measured by the QuantiGene Incubator Temperature Validation Kit thermocouple inserted into the mock Hybridization Plate may differ by 4 C or more QuantiGene Plex 2 0 Reagent System User Manual Page 21 Capturing Target RNA from Total Purified or In Vitro Transcribed RNA Capturing Target RNA from Total Purified or n Vitro Transcribed RNA Procedure To capture target RNA from purified RNA preparations Step Action 1 Prepare the following reagents Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minu
45. of the Filter Plate on a clean dry paper towel d Place the sealed Filter Plate on the Filter Plate Holder and completely wrap the assembly sealed Filter Plate and Filter Plate Holder with aluminum foil e Place on a shaking platform at room temperature and 600 rpm for 30 minutes QuantiGene Plex 2 0 Reagent System User Manual Page 29 Signal Amplification and Detection of RNA Targets Detecting the To detect the signal Signal Step Action 1 Wash away the unbound SAPE a Fill a 100 mL capacity reagent reservoir with SAPE Wash Buffer b Remove the Filter Plate from the shaking platform c Remove the aluminum foil and Plate Seal from the assay wells filter completely then turn off the vacuum d Carefully add 200 uL of SAPE Wash Buffer to each well filter completely then turn off the vacuum e Repeat step 1d for a total of two washes blot the top and bottom of the Filter Plate and return it to the Filter Plate Holder f Immediately proceed to the next step 2 Prepare the plate for analysis on a Luminex instrument a Add 130 uL of SAPE Wash Buffer to each assay well b Very gently reseal the Filter Plate with a foil Plate Seal then blot the bottom of the Filter Plate on a clean dry paper towel c Place the sealed Filter Plate on the Filter Plate Holder then wrap the assembly sealed Filter Plate and Filter Plate Holder in aluminum foil Note At this point the plate can be stored at room te
46. ppendix V Automated Filter Plate Washing Method optional on page 39 for an alternative automated filter plate washing method IMPORTANT This step should be completed within 10 minutes Transfer the overnight hybridization mixture to the Filter Plate and filter a Remove the Hybridization Plate from the shaking incubator and centrifuge at 240 x g for one minute IMPORTANT Adjust temperature of shaking incubator to 50 C 1 C Verify temperature using a QuantiGene Incubator Temperature Validation Kit b Pipet up and down 5 times then completely transfer the hybridization mixture to the Filter Plate c Transfer the Filter Plate to the vacuum manifold and filter d Immediately proceed to the next step Wash away the unbound sample a Carefully add 200 uL of 2 0 Wash Buffer to each of the wells filter completely then turn vacuum off Repeat step 4a two more times for a total of three washes c Blotthe top and bottom of the Filter Plate to completely remove all traces of 2 0 Wash Buffer and return to the Filter Plate Holder d Immediately proceed to the next step QuantiGene Plex 2 0 Reagent System User Manual Signal Amplification and Detection of RNA Targets To hybridize the 2 0 Pre Amplifier continued Step Action 5 Start the 2 0 Pre Amplifier hybridization a Transfer 2 0 Pre Amplifier Working Reagent to a 25 mL capacity reagent reservoir Add 100 uL of 2 0 Pre Amplifier Working
47. protocols to configure the filter plate Setup and vacuum manifold as shown below and adjust the vacuum to 1 2 inches of Hg Operation Note See Appendix V Automated Filter Plate Washing Method optional on page 39 for alternative automated filter plate washing method Filier Pump Protection MultiScreen Pump OCT oe In House Vacuum To verify the performance of the vacuum manifold Step Action 1 Transfer a Filter Plate containing 100 uL of 2 0 Wash Buffer well to the vacuum manifold and firmly press down on all four corners of the plate to ensure a good seal Note Refer to Prepare 2 0 Wash Buffer on page 25 for instructions on preparing 2 0 Wash Buffer Turn on the vacuum Completely filter the solution from all wells 4 Turn off the vacuum Filtration rate should be 5 15 seconds If necessary adjust the pressure IMPORTANT Do not let the Filter Plate dry completely Following filtration the bottom of the Filter Plate should look translucent and not white Filtering too long or at too high a pressure can result in significant Capture Bead loss Luminex Setup Follow the manufacturer s recommended protocol for general operation and and Operation maintenance for your instrument See Appendix II Recommended Maintenance for Luminex Instruments on page 35 for information on cleaning maintenance of your instrument The QuantiGene Plex 2 0 assay is optimized for use at the low sensitivity setti
48. r Manual QuantiGene Plex 2 0 Reagent System Contents and Storage Conditions 2 0 Pre Amplifier E coool 2 0 Amplifier with o K 000 0 t e Dried Blood Spots EE biotinylated Label Probe 0 Whole Blood or LE PAXgene Blood P mRNA IANI NZ BL e FFPE Sections CE A be Animal Tissues ta ban Capture Bead 2 2 Cultured Cells b Step 2 Target RNA Capture Step 3 Signal Amplification Step 4 Detection Step 1 Release Target RNA The sample is analyzed on a Luminex instrument The level of SAPE fluorescence is proportional to the amount of mRNA transcripts captured by the bead a Sequential hybridization of the 2 0 Pre Amplifier 2 0 Amplifier and biotinylated Label Probe respectively for an hour at 50 C Specific mRNA transcripts are captured to their respective beads through a Capture Extender CE Capture Probe CP interaction during an overnight hybridization at 54 C Cells are lysed to release RNA b Binding with Streptavidin conjugated Phycoerythrin SAPE at room temperature for 30 minutes Bio Plex suspension array system or other Luminex based array systems QuantiGene Plex 2 0 Reagent System Contents and Storage Conditions QuantiGene Plex The components of the QuantiGene Plex 2 0 Assay Kit and their recommended Assay Kit storage conditions are listed below The QuantiGene Plex Assay Kit is available in 3 sizes Refer to the product insert for quantities of in
49. ropriate sample processing kit product inserts for detailed procedures and troubleshooting Instrument needle is partially clogged Replace or clean the needle according to the manufacturer s recommendations Bubble introduction into Luminex fluidics Check Luminex probe for proper height then run instrument debubbling protocol Make sure every well contains 130 uL of SAPE Wash Buffer and verify the Luminex sample size is set to 100 uL Using buffers containing precipitates Eliminate precipitates by warming to 37 C for 30 minutes followed by gentle swirling Low Bead Count Troubleshooting low bead count Page 32 Probable Cause Recommended Actions Capture Beads settled or clumped in stock tube Vortex Capture Beads for 30 seconds immediately prior to adding to Working Plex Set Capture Beads were not resuspended prior to transfer to the Filter Plate Pipet up and down to resuspend the Capture Beads in the Hybridization Plate prior to transfer of the hybridization mixture to the Filter Plate Vacuum setting too high Do not exceed a vacuum of 1 2 inches of Hg Vacuum on for too long when removing the buffer from the wells Do not apply vacuum for longer than 15 seconds after the buffer is completely drained from each well of the Filter Plate Filter Plate not shaken enough prior to reading Shake the Filter Plate at 600 rpm for at least two minutes to resuspend the
50. s panomics com Email techsupport panomics com European Headquarters Panomics Srl Via Sardegna 1 20060 Vignate Milano Italy Tel 39 02 95 360 250 Fax 39 02 360 992 Email info_europe panomics com Email order_europe panomics com Email techsupport_europe panomics com Asia Pacific Headquarters Panomics Inc 16F Gemdale Plaza Tower A No 91 Jiango Road Beijing 100022 P R China Tel 86 10 59208157 Fax 86 10 59208111 Email info_asia panomics com Email order_asia panomics com Email techsupport_asia panomics com Contents Section E Introduction 4i iua jx be re al y Eo j k ale la a a RS lala a l l lal di u a ala e By Mc dii 5 About This User Manual lu kk kk kk ka kk kk kk kk n 5 Who Should Read This Manual 0 0 00 KK KK KK KK KIRI eee 5 What This Manual Coves 4 xa soutaire ottia din E eee 5 Safety Warnings and Precautions kk kk KK KK KK KK KK KK KK KK IK KK 5 Contacting PanomicS ase 4 L 3 xa JEW dka 2a s SEE Za QIR ba Ak AW EWA AA n n AA 2A 5 Technical Help gt s5 55 s waw k da e hay UR bh EWAN ad ay welt Abed itat Q 5 About the QuantiGene Plex 2 0 Reagent System 0 0 00 cece KK KK KK 6 Wall Dl N DD oe te Py ee ela hed 6 QuantiGene Plex 2 0 Assay Basics 0 00 kK KK KK KK eee 6 QuantiGene Plex 2 0 Reagent System Contents and Storage Conditions 7 QuantiGene Plex Assay Kit A kk kK KK
51. tes followed by gentle swirling Probe Set and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents to the bottom of the tube Capture Beads Take out of storage right before use and protect from light RNA sample s If frozen thaw on ice Vortex briefly before use Transfer an appropriate volume 5 uL assay well plus 40 overage of Probe Set to a microfuge tube heat to 95 C for 5 minutes using a heat block then place on ice Pre warm Lysis Mixture at 37 C for 30 minutes followed by gentle swirling If appropriate based on expression level of the lowest abundance target or housekeeping RNA in the Plex Set for total RNA or mRNA dilute RNA in nuclease free water so that the desired amount of RNA is present in a volume of 20 uL assay well Use the table below as a guide and scale dilution volumes according to the number of assays to be run IMPORTANT Always verify that assay signals are within both the instrument and assay linear ranges For more information see Optimizing Sample Input on page 9 and Determining Assay Linearity on page 12 Recommended Sample Input Total RNA mRNA ng RNA 2 0 Mag 2 0 Mag copiesper 2 0 Capture Capture 2 0 Capture Capture cell Beads Beads Beads Beads 1 2 500 300 50 6 10 250 30 5 0 6 100 25 0 5 0 06 21000 lt 2 5 lt 0 3 lt 0 05 lt 0 006 For in vitro transcribed IVT RNA
52. tips for each transfer dispense 60 uL of Working Plex Set into each well of the Hybridization Plate IMPORTANT Include 3 wells for assay background control Using a new pipette tip for each transfer add 40 uL tissue homogenate or diluted tissue homogenate to each well of the Hybridization Plate containing Working Plex Set IMPORTANT Add 40 uL of Homogenizing Solution to 3 wells for assay background control Page 20 QuantiGene Plex 2 0 Reagent System User Manual Capturing Target RNA from Fresh Frozen or FFPE Animal Tissue Homogenates To capture target RNA from tissue homogenates continued Step Action 8 Use one of the following methods to seal the plate If using components from a 1 or 3 plate Assay Kit a Tightly seal the Hybridization Plate using the Sealing Mat IMPORTANT Ensure that each individual well of the sealing mat has been snapped into place Failure to completely seal the plate may result in excessive evaporation during the overnight incubation b Cover the plate with the plate lid and secure it with a clamp at each end as shown below Note If clamped plates do not fit in the incubator holder heavy duty rubber bands may be substituted for the clamps Plate Lid Sealing Mat Overnight Hybridization Plate Side View Top View AAA AMM AAAM ALT ERR If using components from a 10 plate Assay Kit seal the Hybridization Plate s with Heat Seali
53. tup and Operation 000 RR RR KRI 14 Luminex Setup and Operation 20 ccc eee eee 14 Validating Luminex Instrument Calibration and Setup 00 15 Capturing Target RNA si Aya balk W aer n kl e rns 15 QuantiGene Plex 2 0 Reagent System User Manual iii Table of Contents About Capturing Target RNA kk kk kk kk kK kK KK KK KK ee 15 Capturing Target RNA from Cultured Cell and Whole Blood Lysates 16 Procedure 2 aces eeu eer deoa ets eee dee EON 16 Capturing Target RNA from Fresh Frozen or FFPE Animal Tissue Homogenates 19 Procedure ecu e pte ERO ERA UAE De PER ARA 19 Capturing Target RNA from Total Purified or In Vitro Transcribed RNA 22 Procedure ote aen rre eu S glo nade Pastas daf Dg ERO tae PE beat Bre s 22 Signal Amplification and Detection of RNA Targets 0000 RR KK KI 25 About this Procedure J kk kk kk kk kk KK KK kK KK KK KK KK es 25 Before You State s42 Ayda Ed eR Ri AE MESI 25 IMPORTANT Precautions kk kk kk KK KK KK KK KK KK KK KK KK KK KK KK 25 Hybridizing the 2 0 Pre Amplifier 0 0 0 0 KK KK KK KK KK KK KI eee 26 Hybridizing the 2 0 Amplifier llle 28 Hybridizing the Label Probe k kk kk kk KK KK KK KK KIR KK KI KK KI KK KK 28 Binding the SAPE 35 3 x ku e SA ee e E ER DES S 29 Detecting the Signal 2 2 2 2 Ea Wa ele ale kala na 30 Section Ill Troubleshooting LAL LAL Ak kk kk
54. wer or more wells scale reagents accordingly Before You Start Bring Amplifier Diluent Label Probe Diluent and SAPE Diluent to room temperature Warm Amplifier Diluent to 37 C for 20 minutes to dissolve any precipitates and mix well by inversion before use Prepare 2 0 Wash Buffer To prepare 2 0 Wash Buffer Step Action 1 Add to a 250 mL graduated cylinder in this order 190 mL nuclease free water 0 6 mL 2 0 Wash Buffer Component 1 10 mL 2 0 Wash Buffer Component 2 Note Scale preparation according to the number of plates to be processed 200 mL is sufficient for processing 1 plate 2 Transfer to a 250 mL bottle and invert to mix Do not store unused 2 0 Wash Buffer Make 2 0 Wash Buffer fresh daily IMPORTANT Precautions Avoid touching the bottom of the Filter Plate with your fingers or benchtop at all times Avoid splashing and cross contamination during all wash steps Do not exceed vacuum pressure of 1 2 inches of Hg during filtration or Capture Bead loss might occur Check Filter Plate for chips and cracks in the bottom Do not use a cracked plate Do not allow Filter Plates to air dry following washes Turn off vacuum as soon as filtration is complete and immediately add the next solution Following the last wash in each series thoroughly blot the top and bottom of the Filter Plate to completely remove all of the Wash Buffer Do not apply pressure to the tops of Filter P
55. x none Alcohol wash 3x 70 ethanol Wash 4x Distilled water Before Shut Down Clean the probe needle before shutting down the instrument as indicated in the following table Step Number of Times Solution Sanitize 2x 2096 bleach Backflush 3x none Alcohol wash 3x 7096 ethanol Wash 4x Distilled water Soak 1x Water QuantiGene Plex 2 0 Reagent System User Manual Page 35 Appendix Ill Common Housekeeping Genes and their Relative Expression Levels Appendix Ill Common Housekeeping Genes and their Relative Expression Levels The table below lists the common housekeeping genes and general guidelines for the linear working range based on the number of cells well These guidelines assume the Luminex instrument has been setup and calibrated as instructed in this user guide We recommend including a minimum of 3 housekeeping genes each at a different relative expression level in all Plex Sets For example include housekeeping genes representing medium high medium and low relative expression levels Housekeeping Genes and Expression Levels Guidelines for cells well Relative Common 2 0 Mag Capture Expression Level Housekeeping Gene 2 0 Capture Beads Beads High ACTB B2M GAPDH 200 10 000 50 5 000 PPIA RPL19 RPL23 RPL32 RPLRO RPS3 RPS18 RPS20 Medium high LDHA PGK1 PPIB 400 80 000 100 30 000 RPL13A UBC Medium HPRT POLR2A 1 600 80 000 400 60 000 T
56. yrene microplate 15 30 C Plate Seals Adhesive backed foil plate sealer 15 30 C Hybridization Plate Assembly 1 and 3 plate Assay Kits 96 well clear polypropylene plate sealing 15 30 C mat and plate lid Plate Clamps 1 and 3 plate Assay Kits Larger binder clips for securing Hybridization 15 30 C Plate Assembly 10 plate Assay Kit Heat Sealable Hybridization 96 well clear polypropylene plate 15 30 C Plate 10 plate Assay Kit Heat Sealing Foil Heat sealing foil plate seal 15 30 C a We recommend storing in an enzyme storage box such as the NEB Cool Box New England Biolabs P N T0400S NEVER store at 80 C In addition to QuantiGene Plex 2 0 Assay Kits two accessory reagents are required to perform QuantiGene 2 0 Plex assays For ordering information please visit our website at www panomics com Accessory Reagent Description Comment QuantiGene Sample Processing Kit Contains reagents and instructions for processing different sample types Specify sample type cultured cells blood samples FFPE samples fresh or frozen animal tissue QuantiGene Plex 2 0 Plex Sets Contains pre pooled target specific 2 0 Probe Set and pre pooled 2 0 Capture Beads or 2 0 Mag Capture Beads Required Reagents and Equipment About Required Materials Equipment and Disposables Page 8 These materials are required to perform the assay but are not supplied

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QuantiGene® Plex 2.0 Reagent System