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1. Order of addition Reagent 1 Well pL 48 Wells uL 96 Wells pL 1 Nuclease free water 3 1 208 417 2 Lysis Mixture 13 7 921 1841 3 Blocking Reagent 2 134 269 4 Proteinase K 0 2 13 27 Capture Beads 1 67 134 Total 20 1343 2688 a Includes 40 overage to enable use of reagent reservoir and multichannel pipettor 9 Vortex Working Bead Mix for 30 seconds to mix then dispense into the Hybridization Plate For fewer than 48 wells 20 QuantiGene Plex DNA Assay User Manual 10 11 12 Using a single channel pipette and a new tip for each transfer dispense 20 uL Working Bead Mix into each well of the Hybridization Plate For 48 wells or more A Using a single channel pipette transfer Working Bead Mix to a 25 mL 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 Bead Mix into each well of the Hybridization Plate Lu IMPORTANT Include 3 wells for assay background control Using a new pipette tip for each transfer add 80 uL neutralized cell lysate to each well of the Hybridization Plate containing Working Bead Mix Lu IMPORTANT Add 80 uL of Background Control prepared in step 5 to 3 wells Seal the Hybridization Plate using a Pressure Seal A Center the Pressure Seal on the Hybridization Plate B Using a soft rubber roller apply firm even pressure across the s
2. ek ritiri h n dk Panda vege ESN base ee NEE 35 Recommended Maintenance for Luminex Instruments 37 About Luminex Maintenance essegi eee eee 37 After Each RUM lt i dw dou ob Bde eden bka d l bude Guu dnd eI id dd S 37 Before ShuL DOWN 2 4 28 23 405 424s Gerda bihaya aside s Putas ee S 37 Blank Plate Maure kk kk kk kk kk GER UR I C UV qui KK KK KK KK KK KK gg 39 Introduction About This User Manual Who This Manual is For This manual is for anyone who has purchased QuantiGene Plex DNA Assay Kit DNA Plex Set and intends to utilize a magnetic plate washer to perform the QuantiGene Plex DNA assay for any of the following sample types u Cultured cells Fresh frozen or formalin fixed paraffin embedded FFPE tissues u Genomic DNA What This Manual Covers This manual provides recommendations and step by step procedures for the following u Assay terminology and data analysis guidelines QuantiGene Plex DNA assay u Troubleshooting Safety Warnings and Precautions All chemicals should be considered potentially hazardous We recommend that this product and 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 Affymetrix Technical Help For technical support contact the appropriate resource provided below base
3. B Using a Misonix 4000 sonicator with microplate horn Misonix 431 MPX amplitude 100 sonicate for 6 minutes Follow manufacturer s instructions to avoid overheating NOTE Shearing will not affect relative fold change A similar result is obtained if you normalize to the normalization gene and reference sample Table 3 8 Effect of Shearing on Relative Sensitivity of the Assay Shearing Method Relative MFI Signal Unsheared 10 Sheared with microplate horn 80 Sheared with single probe sonicator 100 2Unsheared samples refer to samples prepared using the QuantiGene Sample Processing Kit for Cultured Cells 5 For each assay well mix the following reagents a 58 uL cell lysate at 400 cells uL u 5 uL DNA Probe Set a 5 jL 2 5 M NaOH solution For triplicates prepare enough sample for 4 wells to provide overage Include 3 assay wells for background control by replacing cell lysate with Diluted Lysis Mixture 1 volume Lysis Mixture 2 volumes nuclease free water Incubate samples at room temperature for 30 minutes 7 Add 12 uL Neutralization Buffer per assay well Prepare an appropriate volume of Working Bead Mix by combining the following reagents in the order listed Scale according to the number of assays to be run and include appropriate overage Use the table below as a guide Lu IMPORTANT Vortex at maximal speed Capture Beads for 30 seconds before addition Table 3 9 Working Bead Mix
4. ss wees ow ew a dee or CR UE DDR RA RE o a 13 Instrument and Equipment Setup kk kk kk kk kk kk kk KK KK KK KK KI KK KK KK KK K KK KK 13 Target DNA Capture Day 1 kk kk kk kk kk kk kk KK KK KK n 13 Signal Amplification and Detection of Target DNA Day 2 XL kk kk n KK KK KI 13 Instrument and Equipment Setup kk kk kk kk 000 kk kK KK KK KK n 13 Luminex Setup and Operation kk kk kk kk kk kK kK kK KK KK KK KK KK KK KK KK KK KK KK KH 13 Adjusting Luminex Probe Height for Use with Magnetic Separation Plates 14 ii QuantiGeneG Plex DNA Assay User Manual Chapter 4 Appendix A Appendix B Appendix C Appendix D Validating Luminex Instrument Calibration and Setup kk kk KK RR KK KK KK eee 14 Setting up and Validating the Recommended Shaking Incubators 14 Setting Up and Operating the BioTek ELx 405 Magnetic Plate Washer 15 Washing QuantiGene Plex Assay Plates with BioTek ELx 405 Magnetic Plate Washer 17 Capturing Target DNA Day 1 issssssssses tees 18 About Capturing Target DNA xu buka hak n i an dun l k hhh dya d n eae 18 Capturing Target DNA from Cultured Cell Lysate WW kk kK KK KIR KK KI K K KII 18 Capturing Target DNA from Fresh Frozen or FFPE Tissue or Genomic DNA 21 Signal Amplification and Detection of DNA Targets Day 2 iiis isses 24 ABOU TIS PROCSQUEB eee UL EIE Ed a EI NIME 24 Before YOU Start s M MH EH Ste e E
5. Limit of Quantification LOQ LOQ is the lowest MFI that exhibits acceptable accuracy of fold change see Assay Linearity Accuracy of Fold Change on page 7 Assay Linearity Accuracy of Fold Change Assay linearity is defined as all dilutions that exhibit an accuracy of fold change between 80 and 100 To determine assay linearity 1 Run a dilution series of your sample 2 Subtractthe AVG assay background signal from the AVG signal of technical replicates for each target DNA 3 Calculate the ratio of background subtracted AVG MFI from sequential sample dilutions for each target DNA Observed values should be within 20 of the expected ratio of 10096 80 120 NOTE Quantifiable signals are those signals within the assay s linear range 8 QuantiGene Plex DNA Assay User Manual Table 2 6 Ratio of Background Subtracted AVG MFI fir Each Target DNA 3 fold serial Signal background Observed fold Expected fold 96 Obs Exp dilution of the subtracted MFI change change cell lysate pL 60 3100 3 10 3 103 20 1000 2 70 3 90 6 6 370 Guidelines for Assay Optimization Assay Design and Data Analysis Overview This section provides guidelines for the following Optimizing Sample lysis Optimizing Sample input Assay controls Assay replicates Calculation of DNA copy number relative and absolute Optimizing Lysis Conditions To determine optimal sample amount for lysis or homogenization 1 Fol
6. then read immediately F 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 800 rpm for 2 3 minutes then read immediately NOTE See Setting Up Luminex Instrument for QuantiGene Plex DNA Assays on page 35 for setting up the Luminex reader 28 QuantiGene Plex DNA Assay User Manual Troubleshooting Low Assay Signal or Poor Sensitivity Table 4 13 Troubleshooting Low Assay Signal or Poor Performance Probable Cause Recommended Actions Number of DNA target copies below limit of detection Increase the sample input and or perform a recommended method for DNA shearing Incomplete cell lysis and or DNA shearing See Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 Signal amplification reagent is incorrectly prepared Carefully add the correct amounts of DNA Pre Amplifier DNA Amplifier Label Probe and SAPE to the appropriate 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 Magnetic Separation Plate during all incubations Photobleaching of SAPE Protect SAPE from light
7. Incubator Temperature Validation Kit Panomics P N QS0517 4 soft rubber roller Panomics P N QS0515 Assay Terminology and Data Analysis Guidelines Assay Terminology Replicates 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 DNA 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 DNA assay Assay Precision The Coefficient of Variation CV 1s a measure of assay precision QuantiGene Plex DNA Assay CVs are typically less than 15 for technical replicates To determine the assay CV 1 Run technical replicates of each sample 2 Calculate the average signal AVG of technical replicates for each target DNA 3 Calculate the standard deviation SD of signals from technical replicates for each target DNA 4 Calculate the CV CV SD AVG 100 Assay Limit of Detection LOD The LOD is the signal above the background plus 3 standard deviations of the background To calculate assay limit of detection for each target DNA LOD AVG MFI of assay background control wells 3X SD of assay background signals Assay signals below LOD should not be used to draw quantitative conclusions about DNA copy number
8. Kit Shearing can be performed with one of the following methods Method 1 A Transfer samples into 1 5 mL microcentrifuge tubes B Using a Misonix XL 2000 sonicator with a 1 8 inch probe power output 6 sonicate samples for 10 seconds The tip of the probe should be approximately 5 mm below the solution level C Wash the tip between samples by sonicating in sterile distilled water for 10 seconds Method 2 for high throughput shearing with microplate homogenization A Transfer samples into a 96 tube rack Qiagen P N 19566 and 19560 22 QuantiGene Plex DNA Assay User Manual B Using a Misonix 4000 sonicator with microplate horn Misonix 431 MPX amplitude 100 sonicate for 6 minutes Follow manufacturer s instructions to avoid overheating NOTE Shearing will not affect relative fold change A similar result is obtained if you normalize to the normalization gene and reference sample Table 3 10 Effect of Shearing on Relative Sensitivity of the Assay Using Genomic DNA Shearing Method Relative MFI Signal Unsheared 10 Sheared with microplate horn 80 Sheared with single probe sonicator 100 Table 3 11 Effect of Shearing on Relative Sensitivity of the Assay Using Fresh or Frozen Tissue Shearing Method Relative MFI Signal Unsheared 40 Sheared with microplate horn 100 Sheared with single probe sonicator 100 a Unsheared samples refer to samples prepared using the QuantiGene Sample P
9. QuantiGene Plex DNA Assay User Manual To calculate the DNA copy number 1 For each sample determine the average signal MFI for all genes Sample Type Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Background no 4 3 6 3 3 5 sample Reference 59 8 85 8 51 2 52 3 sample Test sample 1 71 7 49 5 127 7 39 Test sample 2 62 92 8 107 5 5 Test sample 3 55 20 52 89 5 For each sample subtract the average background signal for each gene Sample Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Background no 0 0 0 0 sample Reference 55 5 79 5 48 2 47 3 sample Test sample 1 67 4 43 2 124 7 34 Test sample 2 57 7 86 5 104 0 5 Test sample 3 50 7 50 49 84 5 For each sample divide each test gene signal background subtracted by the normalization gene signal background subtracted This will correct for sample preparation sample input and deviations between wells plates and experiments Sample Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Background no na na na na sample Reference 1 1 43 0 87 0 85 sample Test sample 1 1 0 64 1 85 0 5 Test sample 2 1 1 5 1 8 0 01 Test sample 3 1 0 99 0 97 1 67 4 For each test sample divide the normalized signal by the reference sample for each test gene This corrects for the differences of detection efficiency between the test genes in the multi gene analysis Sample
10. and run the Prime step 3 times Creating a Program If the Bio Rad Bio Plex Pro Bio Plex Pro II or Tecan HydroFlex wash station does not have a QGPlex program use the Bio Plex Pro II Hydro Control software to create one To create a program 1 2 Select New program from the File menu and name the file QGPlex Select Plate mode 34 QuantiGene Plex DNA Assay User Manual 3 Set verify plate parameters Go to Plate gt managing plate gt define plate gt select default plate or create new plate gt edit gt plate definition gt plate parameter Table A 1 Plate Parameters Plate Definition Plate Parameter Bio Rad Bio Plex Pro Bio Rad Bio Plex Tecan HydroFlex Wash Station uM Pro Wash Station uM uM Aspiration Y offset 1 2200 1700 1700 Aspiration Y offset 2 2200 2200 2200 Dispense Y offset 2400 2400 2400 Z position bottom 4000 4000 4000 Z position overflow 15000 15000 15000 For units that have the 96 individual magnet configuration For units that have the 9 row magnet configuration Lu IMPORTANT The plate parameter settings provided are a very good starting point however there may be unit to unit variations that may impact performance We have found the most critical settings to be the Aspirate Y offset 1 If residual volume of buffer is greater than 10 pL per well adjust the Aspirate Y offset 1 up or down Add a Cycle step and set to 1 Add a Soak step and set to 1 m
11. below the LOD Data below the LOD should not be used in downstream calculations for drawing quantitative conclusions u Control or reference sample A sample whose DNA copy number has been verified for all targeted genes Normalization gene A target gene with a constant DNA copy number for all assay conditions including the control or reference sample Assay Replicates Run all assay samples with a minimum of duplicates and ideally triplicates Technical replicates are used to calculate assay precision or CV Calculating DNA Copy Number Run the reference sample and test samples on the same plate a Include the normalization gene as a target in the multiplex panel NOTE The QuantiGene Plex assay can resolve 1 copy cell differences ranging from 1 4 copies cell Since the difference of 3 4 copies is 1 4 copy or 25 the average assay CV of 1596 can resolve a 25 difference For 7 8 copies the difference is 1 8 copy or 12 and thus the assay CV must be at least 5 to distinguish 7 copies from 8 copies NOTE If the DNA copy number is not known for a reference sample relative fold changes can be used to estimate the copy number An example is provided to demonstrate how to calculate DNA copy number In this experiment a background reference and 3 samples are tested using a 4 plex panel containing a normalization gene and 3 test genes The example shows how to calculate the DNA copy number for the three test genes 10
12. sU EE greet 24 IMPORTANT Precautions LL kk kK KK KK KK KK eee ee eee ee 24 Hybridizing the DNA Pre Amplifier XA kk kk kk kk kK kK KK KK KK KK KI KK KII K KIR K KK K K KK 24 Hybridizing the DNA Amplifier kk kk kk kk kk kK kK KK KK KK KK KI KK KI KK K K K K KI KK KI KIR KK 25 Hybridizing the Label Probe WW kk kk kk kk kk kK kK KK KK KK KK KK KI KK KI K K K KI KIR KI KIR eee 26 Binding the SAPE 25 odo A o Ela e Rom di n n dk w nS KUD WA Wt lu al SS 26 Detecting the Signal Le abis ERReeRPIS RP bows ERROR ek n 0 0 3 5540 27 Troubleshooting s a uua a quc deae de RO debe E HER e Rom ed e 29 Low Assay Signal or Poor Sensitivity ciis 29 HighiBackgrourg Signal 2 5454 deer biban bda dab We d n b Sk AGIR Kh e an idet nt is 29 low Assay Precision MIG CV iiis s l amp lawa a ke ld EP dues REED eee 30 Low Bead Count 2222 sana cidi bd d y Ray 640444 di a s k 5AQA eid AB El of ded 30 Poor Assay Linearity s a isse as 31 Alternative Magnetic Plate Washers 000 cece eee dd Magnetic Plate Washer Jk kk kk kk kk kk kk KK KK KK KK KK KK KI KK KK KK KK KK KK KK KK KK KH 33 RecommmieridatiOns 5 edocet wy ate dete MIR di dd dan Ki he ad hone teu d eum ey nd 33 Setup and Operaio N siete oie to avers ted tue boe Ged ence Rte Rae 33 Creating a Program 4 tee eee 33 Setting Up Luminex Instrument for QuantiGene Plex DNA Assays 35 Instrument Settings ss i
13. the wash is complete immediately proceed to the next step Hybridize the Label Probe A Transfer the Label Probe Working Reagent to a 25 mL capacity reagent reservoir mon uo Add 100 uL of the Label Probe Working Reagent to each assay well Seal the Magnetic Separation Plate with a foil Plate Seal Shake at 800 rpm for 1 minute at room temperature to ensure beads are resuspended Place the Magnetic Separation Plate into a recommended shaking incubator and incubate for 1 hour at 50 C 1 C and 600 rpm Binding the SAPE To bind SAPE Prepare the SAPE Working Reagent 1 A B C Briefly vortex SAPE to mix then briefly centrifuge to collect the contents at the bottom of the tube Add 36 uL of SAPE to 12 mL of SAPE Diluent Vortex for 15 seconds to mix and protect from light Wash away the unbound Label Probe using the magnetic plate washer A B C Remove the Magnetic Separation Plate from the shaking incubator Remove the foil Plate Seal Place the Magnetic Separation Plate on the plate tray of the plate washer Chapter 3 QuantiGene Plex DNA Assay Procedure 27 D Select the appropriate program E Click Start F When the wash is complete immediately proceed to the next step 3 Bind the SAPE A Transfer the SAPE Working Reagent to a 25 mL capacity reagent reservoir Add 100 uL of the SAPE Working Reagent to each assay well Seal the Magnetic Separation Plate with a foil Plate
14. 2 5 M NaOH solution by adding 1 mL of 10 M NaOH to 3 mL nuclease free water o NAW gt DNA Probe Set and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents at the bottom of the tube Capture Beads Take out of 4 C storage right before use and protect from light Proteinase K Take out of 20 C 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 pipette up and down 5 times then keep at room temperature until use DNA samples If frozen thaw on ice Vortex briefly before use 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 If appropriate dilute genomic DNA in nuclease free water so that the desired amount is present in a volume of 40 uL well Ra IMPORTANT Always verify that assay signals are within both the instrument and assay linear ranges For more information see Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 Shear samples to an average size of 500 base pairs The fragment size of the DNA can be verified by agarose gel electrophoresis NOTE Shearing is not required for FFPE or plant homogenates prepared with Affymetrix Sample Processing
15. 5 For other magnetic plate washers see Alternative Magnetic Plate Washers on page 33 If you are using the hand held magnetic washer for low throughput use refer to the package insert for operating details IMPORTANT Precautions Lu IMPORTANT Avoid splashing and cross contamination during all wash steps Lu IMPORTANT Minimize the exposure of Capture Beads to room light Hybridizing the DNA Pre Amplifier To hybridize the DNA Pre Amplifier 1 Prepare DNA Pre Amplifier Working Reagent A Centrifuge the DNA Pre Amplifier briefly to collect the contents at the bottom of the tube B Add 36 uL of DNA Pre Amplifier to 12 mL of Amplifier Diluent Chapter 3 QuantiGene Plex DNA Assay Procedure 25 C Invert several times to mix Hi IMPORTANT The Amplifier Diluent is a viscous solution Pipette 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 Transfer the overnight hybridization mixture to the Magnetic Separation Plate A Remove the Hybridization Plate from the shaking incubator and centrifuge at 240 x g for one minute E IMPORTANT Adjust temperature of shaking incubator to 50 C 1 C Verify temperature using a QuantiGene Incubator Temperature Validation Kit B Pipette up and down 5 times then completely transfer the hybridization mixture to the Magnetic Separation Plate C Immediate
16. AY Affymetrix User Manual QuantiGene Plex DNA Assay For research use only Not for use in diagnostic procedures Trademarks a Affymetrix and XO are trademarks of Affymetrix Inc QuantiGene is a registered trademark exclusively licensed to Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Citing QuantiGene Plex DNA Reagent Systems in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene Plex DNA Reagent System If a paper cites a QuantiGene Plex DNA Reagent System product and is published in a research journal the lead author s may receive a travel stipend for use at a technology confere
17. Insert for the target bead region associations for your panel 14 QuantiGene Plex DNA Assay User Manual Adjusting Luminex Probe Height for Use with Magnetic Separation Plates If you are running assays on your Luminex instrument that use both Magnetic Separation Plates and Filter Plates it is critical that you verify adjust the probe height for each plate type before reading To adjust the probe height 1 Place 2 x 5 mm discs from the sample needle height align kit into H12 well position of the Affymetrix Magnetic Separation Plate In the needle adjustment setting set the well used for needle adjustment to the H12 position For Bio Plex software the needle adjustment setting will move the plate so that the needle hits the H12 position Adjust the needle as described in the instruction manual provided with your Luminex instrument Validating Luminex Instrument Calibration and Setup Lu IMPORTANT Perform this procedure before you run the assay to ensure the Luminex instrument is setup correctly for your QuantiGene Plex DNA assay To verify the instrument calibration and setup 1 o 0 Mo in bw Setup the Luminex instrument according to the guidelines in Setting Up Luminex Instrument for QuantiGene Plex DNA Assays on page 35 Confirm that the Luminex Probe height is adjusted for use with magnetic separation plates Define a protocol with the appropriate bead regions and set to read 2 wells Lu IMP
18. Luminex P N MCON1 05 or Affymetrix P N PC0623 36 QuantiGeneG Plex DNA Assay User Manual 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 that have rusty or salt deposits should be replaced Luminex P N CN 0007 01 After Each Run Clean the probe needle after each run as indicated in the following table Table C 1 Steps for Cleaning the Probe After Each Run Step Number of Times Solution Sanitize 2x 20 bleach Backflush 3x none Alcohol wash 3x 7096 ethanol Wash 4x Distilled water Before Shut Down Clean the probe needle before shutting down the instrument as indicated in the following table Table C 2 Steps for Cleaning the Probe Needle Step Number of Times Solution Sanitize 2x 2096 bleach Backflush 3x none Alcohol wash 3x 7096 ethanol Wash 4x Distilled water Soak 1x Water 38 QuantiGene Plex DNA Assay User Manual Blank Plate Maps 40 QuantiGene Plex DNA Assay User Manual
19. Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Background no na na na na sample Reference 1 1 1 1 sample Test sample 1 1 0 45 2 13 0 59 Test sample 2 1 1 05 2 08 0 01 Test sample 3 1 0 69 1 11 1 96 Chapter 2 Assay Terminology and Data Analysis Guidelines 11 5 Based on the known copy number of each gene in the reference sample take that number and multiply it by the normalized data in step 4 Sample Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Known copy 2 2 2 1 number of reference sample Reference 2 2 2 1 sample Test sample 1 2 0 89 4 26 0 59 Test sample 2 2 2 09 4 15 0 01 Test sample 3 2 1 38 2 23 1 96 6 Optional Round to nearest whole number to obtain copy number target cell Sample Normalization Gene Test Gene 1 Test Gene 2 Test Gene 3 Known DNA 2 2 2 1 copy number cell of reference sample Reference 2 2 2 1 sample Test sample 1 2 1 4 1 Test sample 2 2 2 4 0 Test sample 3 2 1 2 2 12 QuantiGene Plex DNA Assay User Manual QuantiGene Plex DNA Assay Procedure About the Assay Procedure A general outline of the assay procedure is as follows Sample Preparation Refer to the appropriate QuantiGene Sample Processing Kit Package Insert for sample type specific instructions for preparing cultured cell lysates or tissue homogenates Follow standard laboratory methods for purification of DNA Use samples immediately in QuantiGene or Qua
20. ORTANT Refer to the QuantiGene DNA Plex Set Package Insert for the target bead associations for your panel Vortex Capture Beads at maximum speed for 30 seconds Add 2 5 uL of Capture Beads to 250 uL of SAPE Wash Buffer Vortex to mix Add 100 uL of the Capture Bead mixture into each of 2 wells on the Magnetic Separation Plate Place plate on the magnetic plate washer and perform a total of 15 washes to simulate a real assay Add 130 uL SAPE Wash Buffer per well Shake for 2 minutes at 800 rpm Insert the Magnetic Separation Plate into the instrument and read the 2 wells View the window with the bead regions and DD gate The expected results are u Signals for the expected beads show up on the bead map u Average bead count is greater than 50 region a Single peak in the DD gate window with signals within the set DD gate region Setting up and Validating the Recommended Shaking Incubators This procedure is for the VorTemp 56 incubator holds up to 2 plates To setup the VorTemp 56 incubator and to validate the temperature 1 Place an inverted plate lid into each of the two plate carriers in the VorTemp Shaking Incubator Lu IMPORTANT With the inverted plate lids in place the Vortemp digital display and the actual 2 temperature measured by the QuantiGene Incubator Validation Kit thermocouple inserted into the mock hybridization plate may differ by 4 C or more Set the shaking speed to 600 rpm Chapter 3
21. Plex Pro Pro II and Tecan HydroFlex magnetic plate washers Recommendations Follow the manufacturer s instructions for detailed operating and maintenance procedures For priming prepare at least 40 mL of QuantiGene Plex Wash Buffer and 80 mL of SAPE Wash Buffer for priming Two primes of 40 mL each are required for the SAPE Wash Buffer Use Affymetrix Magnetic Separation Plates Use of alternative plates may negatively impact assay performance Reserve Liquid 1 container connected to channel 1 for QuantiGene Plex Wash Buffer and SAPE Wash Buffer Reserve Liquid 2 container connected to channel 2 for distilled water for rinsing Setup and Operation Setting Up and Operating the Plate Washer 1 2 3 4 9 Add QuantiGene Plex Wash Buffer to Liquid 1 container and distilled water to Liquid 2 container Turn on the magnetic plate washer Run a Rinse followed by Prime step Select the QGPlex program If the magnetic plate washer does not have the QGPlex program see Creating a Program on page 33 Click Start Select the column of sample wells or entire plate to be washed When switching from QuantiGene Plex Wash Buffer to SAPE Wash Buffer put SAPE Wash Buffer in Liquid 1 container and run the Prime step twice After the completion of each wash or at the end of the day run a Rinse step to prevent clogging of the aspirate dispense needles Before shutting down replace Liquid 1 container with distilled water
22. QuantiGene Plex DNA Assay Procedure 15 Set the temperature for the assay Validate the temperature with the QuantiGene Incubator Validation Kit n u If running a single plate place another sealed plate in the second position for balance 6 Validate the temperature at least once per month The following procedure is for the Thermo Scientific MaxQ 4450 Digital Shaking Incubator holds up to 6 plates To setup the Thermo Scientific MaxQ 4450 incubator and to validate the temperature 1 Verify that the plate holders are assembled in the format shown in Figure 3 1 Figure 3 1 Microplate Layout for the Thermo Scientific MaxQ 4550 Set the shaking speed to 300 rpm Set the temperature for the assay Validate the temperature with the QuantiGene Incubator Validation Kit uP wes Validate the temperature at least once per month Setting Up and Operating the BioTek ELx 405 Magnetic Plate Washer Recommendations Follow the manufacturer s instructions for detailed operating and maintenance procedures For priming prepare at least 200 mL each of QuantiGene Plex Wash Buffer and 200 mL of SAPE Wash Buffer u Use Affymetrix Magnetic Separation Plates provided in the QuantiGene Plex DNA Assay Kit and also sold separately Reserve Buffer A container for QuantiGene Plex Wash Buffer and SAPE Wash Buffer Reserve Buffer B container for distilled water for rinsing Hi IMPORTANT Separate purchase of add
23. Seal Completely wrap the Magnetic Separation Plate with aluminum foil mon Ww Place on a shaking platform at room temperature and shake at 800 rpm for 1 minute followed by 600 rpm for 30 minutes Detecting the Signal To detect the signal 1 Wash away the unbound SAPE using the magnetic plate washer A Replace QuantiGene Plex Wash Buffer with SAPE Wash Buffer in Buffer A container Prime with SAPE Wash Buffer Remove the Magnetic Separation Plate from the shaking platform Remove the foil Plate Seal Place the Magnetic Separation Plate on the plate tray of the plate washer Select the appropriate program Click Start When the wash is complete immediately proceed to the next step ze mi mons 2 Prepare the plate for analysis on a Luminex instrument IMPORTANT Verify the probe height in the Luminex instrument is set appropriately for use with Magnetic Separation Plates See Adjusting Luminex Probe Height for Use with Magnetic Separation Plates on page 14 A Add 130 uL of SAPE Wash Buffer to each assay well B Seal the Magnetic Separation Plate with a foil Plate Seal C Wrap the Magnetic Separation Plate in aluminum foil NOTE At this point the plate can be stored at room temperature 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 800 rpm for 2 3 minutes
24. Sonicator XL 2000 1 8 single probe for microcentrifuge tube Misonix P N 431MPX Misonix P N XL 2000 96 tube rack if processing microplates Qiagen P N 19560 and 19566 Microcentrifuge Eppendorf 5415D or equivalent Microplate centrifuge that can achieve 240 x g Eppendorf 5804R and rotor A 2 DWP or equivalent Vortex mixer MLS Nuclease free water MLS Sodium hydroxide pellets Sigma P N 480848 or equivalent Microcentrifuge tubes MLS applications or applications ELx405 Select Magnetic Plate Washer recommended for higher throughput Hand held Magnetic Plate Washer recommended for lower throughput BioTek P N ELx405UCWVSM Affymetrix P N QP0702 temperatures of 50 C 1 C and 54 C 1 C Shaking incubator with microplate adaptor capable of maintaining constant Panomics P N QP0700 120V holds 6 plates or Thermo Scientific MaxQ 4450 Digital holds 6 plates P N SHKE4450 30100 and 30175 6 required or LabNet VorTemp 56 holds 2 plates P N S 0256 Q 6 QuantiGene Plex DNA Assay User Manual Table 1 5 Reagents and Equipment Not Provided Item Source Microplate shaker capable of maintaining a speed of 800 rpm 3 mm orbit Labline model 4625 or equivalent Luminex or Luminex based instrument MiraiBio Bio Rad or other Luminex instrument provider Sample Needle Height Align Kit Luminex P N CN 0015 01 QuantiGene
25. ace it in Buffer A container and repeat steps 4 5 When finished washing place distilled water in Buffer A container and run the PRIME_200 program 3 times 18 QuantiGene Plex DNA Assay User Manual Capturing Target DNA Day 1 About Capturing Target DNA This section provides two protocols for capturing target DNA based on sample type a Cultured cell lysate u Fresh frozen FFPE tissue homogenates or genomic DNA preparations Refer to the appropriate protocol for your sample type Capturing Target DNA from Cultured Cell Lysate bu IMPORTANT Cell lysate must be prepared using the QuantiGene Sample Processing Kit for Cultured Cells To capture target DNA from cultured cell lysates 1 Prepare the following reagents u NaOH El WARNING Explosion hazard Never add water to NaOH pellets May cause burns Use caution Dissolution of NaOH in water is exothermic Prepare 10 M NaOH by slowly dissolving 4 g of NaOH pellets in 6 mL nuclease free water Adjust the volume to 10 mL using nuclease free water Store at room temperature good for up to 1 year Weekly prepare 2 5 M NaOH solution by adding 1 mL of 10 M NaOH to 3 mL nuclease free water DNA Probe Set and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents at the bottom of the tube o n wg gt a Capture Beads Take out of 4 C storage right before use and protect from light a Proteinase K Tak
26. adjust the DD gate around the largest peak which represents the singlet beads Adjustments can be made during the processing of the first sample Lu 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 Table B 1 Luminex Instrument Settings Software Instrument Type of Bead Calibration DD gate Sample Size Timeout Bead Events Platform and Supplier uL sec Bead region xPonent Luminex v3 0 Magnetic PROTOCOL Automatically set 100 40 100 BEAD TYPE select magnetic 1S100 Luminex v2 3 Standard 5 000 25 000 100 40 100 NEW BATCH gt CREATE ASSAY TEMPLATE gt Acq Detail enter DD gate value MasterPlex CT MiraiBio Standard ACQUISITION 5 000 25 000 100 40 100 v1 0 SET UP gt SET UP enter DD gate value MasterPlex CT MiraiBio Magnetic ACQUISITION Automatically set 100 40 100 v1 2 TEMPLATE SET UP gt BEAD TYPE select Magnetic Bio Plex 4 0 Bio Rad Standard 5 000 25 000 100 45 100 RUN PROTOCOL gt ADVANCE WINDOW SETTING enter DD gate values Bio Plex 5 0 Bio Rad Standard Automatically set 100 45 100 SELECT ANALYTE gt EDIT PANEL select MagFlex Applied Station Applied Standard TEMPLATE SET 5 000 25 000 100 40 100 Cytometry UP gt enter DD gate value You must use MagPex calibrator beads for calibration MagPlex Calibration Beads Luminex P N MCAL1 05 or Affymetrix P N PC0622 and MagPlex Control Beads
27. am This links the SOAK 01 to the WASH 01 program to create the QuantiGene Plex wash program Go to the LED window on the washer and follow the sequence Main Menu gt Define gt Create gt More gt Link gt Enter SOAK 01 gt Enter WASH 01 F IMPORTANT The wash program settings provided are a very good starting point however there can be unit to unit variations that may impact performance We have found the most critical settings to be the Horizontal Aspiration Position and the Horizontal Y Aspiration Position If residual volume of buffer is greater than 10 pL per well for a 96 well plate adjust Horizontal Aspiration Position and the Horizontal Y Aspiration Position Chapter 3 QuantiGene Plex DNA Assay Procedure 17 Washing QuantiGene Plex Assay Plates with BioTek ELx 405 Magnetic Plate Washer To wash plates 1 2 Turn on the plate washer Run a RINSE followed by a PRIME 200 program with distilled water in Buffer B container A To RINSE go to Main Menu gt Run gt Maint gt Day_Rinse gt Rinse B To PRIME go to Main Menu gt Run gt Prime gt Prime_200 default set by the manufacturer Place QuantiGene Wash Buffer in Buffer A container Run another PRIME_200 program Select the QuantiGene Plex wash program Go to Main Menu gt More gt LINK 01 as defined in Creating a Wash Program on page 16 Place Magnetic Separation Plate on top of the plate holder and press Start When switching to SAPE Wash Buffer pl
28. cedure for determining optimal lysis test different sonication settings for shearing DNA in cell tissue or genomic DNA samples For genomic DNA the optimal DNA fragment size is 500 base pairs as verified by gel electrophoresis Optimizing Sample Input for QuantiGene Plex DNA Assay After you have determined the optimal lysis conditions for sample preparation use the following guidelines to determine the optimal sample amount to use for the QuantiGene Plex DNA assay Resulting signal from the sample is above the LOQ The LOQ is 20 000 DNA copies well Amount of sample is high enough to compensate for sample loading error For example if the amount of loaded sample can deviate more than 4 times then increase the sample input by 4 to ensure detection a If the amount of sample is not limiting use an input that has a signal background ratio of at least 10 fold Background is defined as signal from a sample well that has contains no sample Resulting signal from the sample is around 100 mean fluorescent intensity MFI The Luminex 200 exhibits a range of 2 20 000 MFI at low PMT settings Hence 100 MFI should be sufficient to determine DNA copy number variation from 1 100 copies cell Assay Controls All experiments must have the following controls for determining DNA copy number u Assay background A sample well that contains all the assay components except for the sample The background control is also used to determine data that is
29. correct setup and operation of recommend shaking incubators refer to Setting up and Validating the Recommended Shaking Incubators on page 14 24 QuantiGene Plex DNA Assay User Manual Signal Amplification and Detection of DNA Targets Day 2 About this Procedure These instructions are for processing one 96 well plate using multichannel pipettes reagent reservoirs and a magnetic plate washer To process fewer 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 Wash Buffer To prepare Wash Buffer 1 Add to a 250 mL graduated cylinder in this order 150 mL nuclease free water a 0 6 mL Wash Buffer Component 1 a 10 mL Wash Buffer Component 2 Bring the volume to 200 mL with nuclease free water NOTE Scale preparation according to the number of plates to be processed and the prime volume required for setup of the operation of the magnetic plate washer For each plate plan for 160 mL Wash Buffer this does not include priming volume 2 Transfer to a 250 mL bottle and invert to mix Do not store unused Wash Buffer Make Wash Buffer fresh daily Prepare Magnetic Plate Washer Prepare the BioTek Magnetic Plate washer as described in Setting Up and Operating the BioTek ELx 405 Magnetic Plate Washer on page 1
30. ct 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 fluidics Bubble introduction into Luminex 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 Linearity Chapter 4 Troubleshooting 31 Table 4 17 Troubleshooting Poor Assay Linearity Probable Cause Recommended Actions Incomplete cell lysis See Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 Inadequate sample preparation Refer to the appropriate sample processing kit product inserts for detailed procedures and troubleshooting Instrument saturation Signals gt 20 000 MFI on Luminex instruments are saturated Assay saturation Perform serial dilution of sample to ensure appropriate fold change is observed See Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 for more information 32 QuantiGeneG Plex DNA Assay User Manual Alternative Magnetic Plate Washers Magnetic Plate Washer This appendix provides information for the Bio Rad Bio
31. d on your geographical location For an updated list of FAQs and product support literature visit our website at WWW panomics com Table 1 1 Contacting Affymetrix Location Contact Information North America 1 877 726 6642 option 3 pqbhelp affymetrix com Europe 39 02 95 360 250 techsupport_europe affymetrix com Asia 86 10 59208157 techsupport_asia affymetrix com 2 QuantiGene Plex DNA Assay User Manual About the QuantiGene Plex DNA Reagent System The QuantiGene Plex DNA Reagent System consists of 2 3 modules each sold separately QuantiGene Plex DNA Assay Kit Contains the generic reagents plates and seals required for running the assay QuantiGene DNA Plex Set Contains pooled Probe Set for specified targets and associated magnetic Capture Beads for running the assay u QuantiGene Sample Processing Kit Contains two reagents and a procedure for release and stabilization of sample DNA and RNA for use in QuantiGene assays This kit is not required if working with genomic DNA samples Introduction The QuantiGene Plex DNA Reagent System is designed to quantitate in a single well multiple target specific DNA molecules copy number variation present in a Cultured cell lysates u Fresh or frozen tissue homogenates FFPE animal tissue homogenates Genomic DNA Please refer to the QuantiGene Sample Processing Kit Package Insert for instructions on preparing cultured cell lysates or tissue h
32. e out of 20 C storage right before use and place on ice a Cultured cell lysates If frozen 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 Lu IMPORTANT Do not put samples back on ice Pre warm Lysis Mixture at 37 C for 30 minutes followed by gentle swirling If appropriate dilute cell lysates with diluted Lysis Mixture 1 volume Lysis Mixture plus 2 volumes nuclease free water so that the desired amount of sample is present in 58 nL well For triplicates with overage prepare 260 uL sample Hi IMPORTANT Always verify that assay signals are within both the instrument and assay linear ranges For more information see Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 4 Shear samples with one of the following methods Method 1 A Transfer 260 uL of sample into 1 5 mL microcentrifuge tube Chapter 3 QuantiGene Plex DNA Assay Procedure 19 B Using a Misonix XL 2000 sonicator with a 1 8 inch probe power output 6 sonicate samples for 10 seconds The tip of the probe should be approximately 5 mm below the solution level C Wash the tip between samples by sonicating in sterile distilled water for 10 seconds Method 2 for high throughput shearing with microplate homogenization A Transfer samples into a 96 tube rack Qiagen P N 19566 and 19560
33. eal Alternatively if using Heat Sealing Foils purchased separately Affymetrix P N QP0542 and an ABgene heat sealer ABGene AB 0384or AB 0563 1000 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 the Hybridization Plate into the shaking incubator Incubate for 18 22 hours at 54 C 1 C and appropriate shaking rpm Lu IMPORTANT For correct setup and operation of recommend shaking incubators refer to Setting up and Validating the Recommended Shaking Incubators on page 14 Chapter 3 QuantiGene Plex DNA Assay Procedure 21 Capturing Target DNA from Fresh Frozen or FFPE Tissue or Genomic DNA E IMPORTANT Tissue homogenates must be prepared using the QuantiGene Sample Processing Kit for tissue To capture target DNA from tissue homogenates or genomic DNA 1 Prepare the following reagents u NaOH n WARNING Explosion hazard Never add water to NaOH pellets May cause burns Use caution Dissolution of NaOH in water is exothermic Prepare 10 M NaOH by slowly dissolving 4 g of NaOH pellets in 6 mL nuclease free water Adjust the volume to 10 mL using nuclease free water Store at room temperature good for up to 1 year Weekly prepare
34. escent dyed microspheres beads lasers and digital signal processing to allow multiplexing of up to 100 unique assays within a single sample The QuantiGene Plex DNA assay is compatible with all Luminex based instruments currently available Assay Principal The QuantiGene Plex DNA assay utilizes fluorescent microspheres Capture Beads as a support to capture specific DNA molecules The ability to quantify multiple target specific DNA molecules in a single sample lies in the design of the Probe Sets For each DNA 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 DNA is provided The CEs discriminate among the different Capture Beads within the bead array while capturing via cooperative hybridization the target DNA 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 DNA molecules present in the sample 0000 paas l DNA Pre Amplifier sss lr ed DNA Ampl
35. ifier with biotinylated Label Probe pe wu Streptavidin Phycoerythrin Ab Plant Tissues 4 DNA IVR BL eem pene o J FFPE Sections CE 2 a e AWRY EON I 4x Animal Tissues Sa Capture pem Bead Cultured Cells Step 1 Release Target DNA Step 2 Target DNA Capture Step 3 Signal Amplification Step 4 Detection a Sequential hybridization of the DNA Pre Amplifier and DNA Amplifier and biotinylated Label Probe respectively for an hour at 50 C Cells are lysed to release DNA DNA is sheared if necessary and denatured Specific DNA fragments are captured to their respective beads through a Capture Extender CE Capture Probe CP interaction during an overnight hybridization at 54 C b Binding with Streptavidin conjugated Phycoerythrin SAPE at room temperature for 30 minutes The sample is analyzed ona Luminex instrument The level of SAPE fluorescence is proportional to the amount of DNA targets captured by the bead Bio Plex suspension array system or other Luminex based array systems 4 QuantiGene Plex DNA Assay User Manual QuantiGene Plex DNA Reagent System Contents and Storage Conditions QuantiGene Plex DNA Assay Kit The components of the QuantiGene Plex DNA Assay Kit and their recommended storage conditions are listed below The QuantiGene Plex DNA Assay Kit is designed for use with magnetic plate washers and is available in 3 sizes Refer to the package i
36. inute 10 sec Add an Aspirate step and set Mode Normal a Z Position custom 4 2 mm u Time 2s Head speed 10 mm s u Aspirate rate 7 Adda Cycle step and set to 3 Add a Dispense step and set a Z Position Overflow u Volume 100 uL u Channel 1 Dispension rate 300 ul s 9 Add a Soak step and set to 20 sec 10 Add an Aspirate step and set Mode Normal a Z Position custom 4 2 mm a Time Is Head speed 10 mm s Aspirate rate 1 For a small number of units sold before November 2008 designated Series A the 4 2 mm setting should be changed to 5 0 mm If you are unsure please contact your Bio Rad representative Setting Up Luminex Instrument for QuantiGene Plex DNA Assays Instrument Settings Calibrate the instrument using the standard PMT setting may be referred to as low by some suppliers The key information in this table is related to the bead calibration required and setting of the DD gate Because of continual software updates we recommend that you contact your Luminex instrument supplier and verify the latest recommendations for setup to 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
37. itional SAPE Wash Buffer and Wash Buffer components will be necessary to accommodate the larger priming volume required for the BioTek Elx 405 magnetic plate washer 16 QuantiGene Plex DNA Assay User Manual Creating a Wash Program E NOTE Instructions on how to create wash soak and link programs can also be found in the BioTek ELx 405 Magnetic Plate Washer user guide IMPORTANT Always follow the vendors update procedures for creating a magnetic plate wash program To create a wash program 1 Create SOAK 01 program Go to the LED window on the washer and follow the sequence Main Menu Define gt Create gt Soak gt Select Soak Program gt Enter Program Name gt 60 Seconds Create WASH 01 program Go to the LED window on the washer and follow the sequence Main Menu gt Define Create gt Wash gt Select Wash Program gt Enter Program Name gt Method Dispense Aspirate and then enter then following data Wash buffer buffer A u Plate type 96 u Cycles 3 u Soak 15 sec Dispense volume 100 pL Dispense flow rate 1 Dispense height 120 Horizontal dispense position 30 Horizontal Y dispense position 00 a Aspirate height 46 Horizontal aspirate position 45 Horizontal Y aspirate position 10 u Aspirate rate 01 Aspiration delay 0000 msec Crosswise aspiration No Final aspiration Yes a Aspiration delay 0000 msec Create LINK 01 progr
38. itions 4 QuantiGene Plex DNA Assay Kit esae ap E IU ER a wee ee eae 2 4 Required Reagents and Equipment Not Provided llle 5 Reagents and Equipment 3 4iL ttti urbe wed au EQUO ERES ERN RE dq 5 Assay Terminology and Data Analysis Guidelines H X k 7 Assay Terminology gt 5 ehe pebereqdepadoeebdbpdopeRPpPURet ne desde bor Puer de debet 7 Replicdtes x edet ee E EHE uo eed uhi pe mm m 7 Assay Precision lt a 4 ss anye W n h b ek l P a k dL und yl eet b PP 7 Assay Limit of Detection LOD 2 sa an sa da la a la had ad n P eR PRI aes 7 Limit oF Quanittication OQ i gt i way ELM 4A k n FERE bd ll y k ha oe ab 7 Assay Linearity Accuracy of Fold Change 0 0 cc a 7 Guidelines for Assay Optimization Assay Design and Data Analysis 8 eri dee EEKEKRRRrrR aer a r daa Ages hy Sohne ao Soe Bh rn 8 Optimizing EysisCOnditlons gt ks hee conta la e pt Rede eh e E CO I GRO RR Frasi da 8 Optimizing Sample Input for QuantiGene Plex DNA Assay kK K KIRR K K KI 9 Assay CONOS s s de ed POR Wj D 2 eens a dede a d poc qe e FG Be ce 9 Assay RepliCates 12 eed ded AA RA deb nt wed Led eeu iii ue PET 9 Calculating DNA Copy Number kk kk kk kk kk kK KK s 9 QuantiGene Plex DNA Assay Procedure kk kk kk KK KK RR KK KK K 13 About the Assay Procedure lt stana dnd alla eere 13 Sample Preparation
39. ix into each well of the Hybridization Plate For 48 wells or more A Using a single channel pipette transfer Working Bead Mix to a 25 mL 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 Bead Mix into each well of the Hybridization Plate Lu IMPORTANT Include 3 wells for assay background control Using a new pipette tip for each transfer add 80 uL neutralized tissue homogenate or genomic DNA to each well of the Hybridization Plate containing Working Bead Mix Lu IMPORTANT Add 80 pL of Background Control prepared in step 5 to 3 wells Seal the Hybridization Plate using a Pressure Seal A Center the Pressure Seal on the Hybridization Plate B Using a soft rubber roller apply firm even pressure across the seal Alternatively if using Heat Sealing Foils purchased separately Affymetrix P N QP0542 and an ABgene heat sealer ABGene AB 0384or AB 0563 1000 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 the Hybridization Plate into the shaking incubator Incubate for 18 22 hours at 54 C 1 C and appropriate shaking rpm IMPORTANT For
40. late 96 well clear polypropylene plate 15 30 C Pressure Seals Clear pressure activated seal for use with the overnight 15 30 C hybridization plate We recommend storing in an enzyme storage box such as the NEB Cool Box New England Biolabs P N TO400S NEVER store at 80 C Accessory Reagents Chapter 1 Introduction 5 In addition to QuantiGene Plex DNA Assay Kits 1 2 additional assay modules are required to perform QuantiGene Plex DNA assays For ordering information please visit our website at www panomics com Table 1 4 Accessory Reagents Accessory Reagent Description Comment QuantiGene Sample Contains reagents and procedures for processing different sample types Specify Processing Kit sample type cultured cells FFPE samples fresh or frozen tissue NOTE This kit is not required if working with genomic DNA QuantiGene DNA Plex Set Contains pooled target specific DNA Probe Set and pooled magnetic Capture Beads Required Reagents and Equipment Not Provided Reagents and Equipment Table 1 5 Reagents and Equipment Not Provided Item Source 20 uL 20 200 uL and 200 1 000 uL Adjustable single and multi channel precision pipettes for dispensing 1 Major Laboratory Supplier MLS Reagent reservoirs 25 mL capacity 100 mL capacity VWR P N 89094 662 or equivalent Corning Costar P N CLS 4873 or equivalent Sonicator 4000 for processing microplates or
41. low the recommended amount of cell number or tissue amount per volume of lysis mixture solution or homogenization solution listed in the Sample Processing Kit package insert for the specific sample types Recommendations are summarized below To ensure optimal lysis in the initial experiment run a test range as indicated in the table Table 2 7 Recommended sample amount for preparation Cultured Cells Tissue Recommended 400 cells uL Working Lysis Mixture 5 mg 300 uL Working Tissue Homogenization Solution Test Range 200 400 800 cells uL Working Lysis 2 5 5 0 10 mg 300 uL Working Mixture Tissue Homogenization Solution 2 For each lysate prepare a 3 fold serial dilution to determine the assay performance Assay performance is determined by calculating the following u LOD u LOQ Assay linearity assay CV Calculate the assay performance for each sample to determine which one had the best performance and use that amount of cells or tissue for future experiments Incomplete or poor lysis will produce high assay CV poor linearity and poor LOD for all analytes in the plex panel To determine the optimal lysis method for a sample type Following the procedure for determining optimal lysis test different lysis methods for example Tissue lyser or liquid nitrogen Chapter 2 Assay Terminology and Data Analysis Guidelines 9 To determine the optimal sonication conditions for shearing DNA Following the pro
42. ly proceed to the next step 3 Wash away the unbound sample using the magnetic plate washer A Place the Magnetic Separation Plate on the plate holder of the plate washer B Select the appropriate program C Click Start D When the wash is complete immediately proceed to the next step IMPORTANT Wash program takes approximately 5 minutes for an entire plate Do not allow plate to dry as this will result in high background 4 Start the DNA Pre Amplifier hybridization A Transfer DNA Pre Amplifier Working Reagent to a 25 mL capacity reagent reservoir Add 100 uL of DNA Pre Amplifier Working Reagent to each assay well Seal the Magnetic Separation Plate with a foil Plate Seal Shake at 800 rpm for 1 minute at room temperature to ensure beads are resuspended mon Ww Place the Magnetic Separation Plate into a recommended shaking incubator and incubate for 1 hour at 50 C 1 C and appropriate shaking rpm Lu IMPORTANT For correct setup and operation of recommend shaking incubators refer to Setting up and Validating the Recommended Shaking Incubators on page 14 Hybridizing the DNA Amplifier To hybridize the DNA Amplifier 1 Prepare DNA Amplifier Working Reagent A Centrifuge the DNA Amplifier briefly to collect the contents at the bottom of the tube B Add 36 uL of DNA Amplifier to 12 mL of Amplifier Diluent C Invert several times to mix 2 Wash away the unbound DNA Pre Amplifier using the magne
43. nce or tradeshow by sending a copy of the paper to our technical support group at pqbhelp affymetrix com or via fax at 510 818 2610 Disclaimer Affymetrix 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 Affymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2009 Affymetrix Inc All rights reserved Contents Introductions eere reee a a TCU TURNIER 1 Chapter 1 Chapter 2 Chapter 3 About This User Manual rece ro Op oc REC ODE oe DR de Eod 1 Who This Manual is FOF si gt sss Aa al ee Aa Bord alay a RR Rue EE SE dot cios 1 What This Manual COV6ts sg huk kalan n e P dee Ip ERU VU CAR ROC Ed 1 Safety Warnings and Precautions j jj s 4 4z 144 55 4402 4535 s 1 Contacting ATIVImett X uuo oe RR onore e m n 1 Technical Helo cae aa DD re Ro Saca HH HHHH HHHH 1 About the QuantiGene Plex DNA Reagent System 0 0 0 0 KK KK lisse 2 auigero Nalo D N O ETT DUST a e E A E A E a E e Gei 2 QuantiGene Plex DNA Assay Specifications 2 kk kk KK KK KK KK KK eee 2 QuantiGene Plex DNA Assay Basics kk a kk kk kK kK KK KI KK KI KI KK KIRI KI KIRI KI KI KK KIR KI KK IK 2 QuantiGene Plex DNA Reagent System Contents and Storage Cond
44. nsert for quantities of individual components supplied Kit components have a shelf life of 6 months from the date of receipt Each QuantiGene Plex DNA Assay Kit is supplied in 3 separate parts based on storage temperature Table 1 3 QuantiGene Plex DNA Assay Kit Components and Storage Conditions Component Description Storage Proteinase K Proteinase K in aqueous buffered solution 20 C Blocking Reagent Aqueous buffered solution containing a preservative 20 C Label Probe Biotinylated oligonucleotide in aqueous buffered solution 20 C DNA Pre Amplifier DNA in aqueous buffered solution 20 C DNA Amplifier DNA in aqueous buffered solution 20 C Amplifier Diluent Aqueous buffered solution containing protein and a preservative 2 8 C Label Probe Diluent Aqueous buffered solution containing protein and a preservative 2 8 C SAPE Streptavidin conjugated R Phycoerythrin 2 8 C SAPE Diluent Aqueous buffered solution containing protein and a preservative 2 8 C Lysis Mixture Aqueous buffered solution containing a preservative 15 30 C Neutralization Buffer Aqueous buffered solution 15 30 C Wash Buffer Component 1 Aqueous solution 15 30 C Wash Buffer Component 2 Aqueous buffered solution 15 30 C SAPE Wash Buffer Aqueous buffered solution 15 30 C Magnetic Separation Plates 96 well microplate 15 30 C Plate Seals Adhesive backed foil plate sealer 15 30 C Hybridization P
45. ntiGene Plex assays or store at 80 C until use Instrument and Equipment Setup u Setup Luminex instrument u Validate Luminex instrument setup and operation u Validate shaking incubator setup and operation Setup and operation of BioTek ELx 405 Magnetic Plate Washer Target DNA Capture Day 1 a Dilute samples if appropriate Sonicate samples to fragment DNA Denature samples in the presence of DNA Probe Set Prepare Working Bead Mix Dispense Working Bead Mix samples and controls into Hybridization Plate Hybridize samples overnight Signal Amplification and Detection of Target DNA Day 2 u Transfer samples to Magnetic Separation Plate and wash away unbound material u Sequentially hybridize the DNA Pre Amplifier DNA Amplifier Label Probe and SAPE Analyze samples using a Luminex based instrument Instrument and Equipment Setup Luminex Setup and Operation Follow the manufacturer s recommended protocol for general operation and maintenance for your instrument See Alternative Magnetic Plate Washers on page 33 for information on cleaning maintenance of your instrument The QuantiGene Plex DNA assay is optimized for use at the low sensitivity setting The sensitivity setting is performed during calibration Refer to the table in Setting Up Luminex Instrument for QuantiGene Plex DNA Assays on page 35 for information on setting up your Luminex instrument Lu IMPORTANT Refer to the QuantiGene DNA Plex Set Product
46. omogenates To prepare DNA follow standard laboratory methods QuantiGene Plex DNA Assay Specifications Table 1 2 QuantiGene Plex DNA Assay Specifications Item Value Limit of Detection LOD 10 000 copies well Limit of Quantitation LOQ 20 000 copies well Linearity 2 5 logs Plex Level 3 34 plex Sample Types Cultured cells fresh frozen tissue plant or animal FFPE Tissues Plate Format 96 well Automation Compatibility Yese Defined as signal greater than background plus three standard deviations of the background Defined as the signal just above the lowest signal that obtains an 80 120 spike recovery Defined as the assay window that consistently achieves a 80 120 accuracy of fold change 4 Contact Affymetrix for information on a 384 well version Batch or full automation using standard automation equipment Contact Technical Support for protocol details and assistance in set ting up your automation system QuantiGene Plex DNA Assay Basics Assay Technology The QuantiGene Plex DNA assay combines branched DNA bDNA signal amplification and multi analyte profiling beads xMAP technologies to enable the detection and quantitation of multiple DNA targets simultaneously The bDNA assay is a hybridization based method of target specific DNA quantitation that amplifies signal rather than target DNA Chapter 1 Introduction 3 The xMAP system combines a flow cytometer fluor
47. rocessing Kit for Tissues For each assay well mix the following reagents u 40 uL sample u 18 uL Lysis Mixture 5 uL Probe Set u 5 pL 2 5 M NaOH solution For triplicates prepare enough sample for 4 wells to provide overage Include 3 assay wells for background control by replacing tissue homogenate with homogenizing solution or water if analyzing purified genomic DNA Incubate at room temperature for 30 minutes Add 12 uL Neutralization Buffer per assay well Prepare an appropriate volume of Working Bead Mix by combining the following reagents in the order listed Scale according to the number of assays to be run and include appropriate overage Use the table below as a guide Lu IMPORTANT Vortex at maximal speed Capture Beads for 30 seconds before addition Table 3 12 Working Bead Mix Order of addition Reagent 1 Well pL 48 Wells uL 96 Wells pL 1 Nuclease free water 1 8 121 242 2 Lysis Mixture 15 1008 2016 3 Blocking Reagent 2 134 268 4 Proteinase K 0 2 13 27 5 Capture Beads 1 67 134 Total 20 1343 2687 a Includes 40 overage to enable use of reagent reservoir and multichannel pipettor 9 Vortex Working Bead Mix for 30 seconds to mix then dispense into the Hybridization Plate 10 11 12 Chapter 3 QuantiGene Plex DNA Assay Procedure 23 For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 20 uL Working Bead M
48. s then transfer supernatants to a new tube and repeat centrifugation and transfer step before use Incomplete cell lysis See Guidelines for Assay Optimization Assay Design and Data Analysis on page 8 Instrument needle is partially clogged Replace or clean the needle according to the manufacturer s recommendations fluidics Bubble introduction into Luminex 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 Low Bead Count Using buffers containing precipitates Eliminate precipitates by warming to 37 C for 30 minutes followed by gentle swirling If precipitate remains continue with the incubation Table 4 16 Troubleshooting Low Bead Count Probable Cause Recommended Actions stock tube Capture Beads settled or clumped in Vortex Capture Beads for 30 seconds immediately prior to adding to Working Bead Mix Separation Plate Capture Beads were not resuspended prior to transfer to the Magnetic Pipette up and down to resuspend the Capture Beads in the Hybridization Plate prior to transfer of the hybridization mixture to the Magnetic Separation Plate Magnetic Separation Plate not shaken enough prior to reading Shake the Magnetic Separation Plate at 800 rpm for at least two minutes to resuspend the beads before reading the plate Incorre
49. 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 Significant DNA degradation Run a 1 agarose gel to verify DNA integrity Incomplete DNA denaturation High Background Signal Use freshly prepared NaOH Table 4 14 Troubleshooting High Background Signal Probable Cause Recommended Actions Sub optimal assay conditions Follow the recommended incubation times and temperature Shake the Magnetic Separation Plate during all incubations Poor washing Set up the magnetic washer with 5 10 uL of residual volume for each wash step Verify washing program on the magnetic washer 30 QuantiGene Plex DNA Assay User Manual Low Assay Precision High CV Table 4 15 Troubleshooting Low Assay Precision High CV Probable Cause Recommended Actions Inaccurate pipetting Use only calibrated precision pipettes Affix tips securely Use a new tip for each transfer Pipette slowly and carefully avoiding bubbles Residual Wash Buffer Set the magnetic washer so that there is only 5 10 uL of residual volume for each wash step 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 minute
50. tic plate washer A Remove the Magnetic Separation Plate from the shaking incubator B Remove the foil Plate Seal C Place the Magnetic Separation Plate on the plate tray of the plate washer D Select the appropriate program 26 QuantiGene Plex DNA Assay User Manual E F Click Start When the wash is complete immediately proceed to the next step 3 Start the DNA Amplifier hybridization A Add 100 uL of DNA Amplifier Working Reagent to each assay well Seal the Magnetic Separation Plate with a foil Plate Seal mon uou Transfer DNA Amplifier Working Reagent to a 25 mL capacity reagent reservoir Shake at 800 rpm for 1 minute at room temperature to ensure beads are resuspended Place the Magnetic Separation Plate into a recommended shaking incubator and incubate for 1 hour at 50 C 1 C and 600 rpm Hybridizing the Label Probe To hybridize the Label Probe 1 Prepare the Label Probe Working Reagent A B C Centrifuge Label Probe briefly to collect the contents at the bottom of the tube Add 36 uL of Label Probe to 12 mL of Label Probe Diluent Vortex for 15 seconds to mix Wash away the unbound DNA Amplifier using the magnetic plate washer mn m on gg gt Remove the Magnetic Separation Plate from the shaking incubator Remove the foil Plate Seal Place the Magnetic Separation Plate on the plate tray of the plate washer Select the appropriate program Click Start When

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