LM DNA Kit Product Insert
Contents
1. ccc ece eee eeeeeeenneeeeeeeeenneaeens 6 Specimen Collection and Preparation 3 Limited LICENSEC ccccceeeeeeeeeeeeeeeeeenneees 6 PPOCC GUNG cc scans nas suesnesscvedsescasmmevetunsceens 3 Trademarks USed ccccceeeeeeeeeeneeeeneenes 7 A Materials Provided 000008 3 Appendix Aiscacesceivessiennesetvessievassedvssviensaieds 7 B Materials Reagents and Equipment Gel Electrophoresis cceeceeeeeeees 7 Required but not Provided 3 Gel Interpretation ccceeeeeee eee e eens 7 DEFINITION OF SYMBOLS Product Labels and Supplemental Documents Temperature Upper limit of limitations temperature Q Sufficient for N EEPE NA Tests Caution Consult See instructions Manufacturer Danger ops Instructions foruse for Use Catalog Batch Code LOT nee Use By Keep away Date from light Page 1 of 7 LC880RUO 12 05 15 REAGENTS BY CATALOG NUMBER LIFECODES KIR Typing Kit for use with Luminex Product 545110R Product Number Fill volume MX K1 LIFECODES KIR 1 Master Mix 545101 170 uL MX K2 LIFECODES KIR 2 Master Mix 545201 170 uL 20 ix 2 8 C Sufficient for 20 LIFECODES KIR Probe Mix 545303 720 uL Protect From Light ulticient tor ZU samples Ds Dilution Solution 628155 18 30 C Probe Mixes are light sensitive keep exposure to light at a minimum CAUTION Do not use components past their expiration date SUMMARY2. Caution e There is a separate threshold table for each locus e These threshold tables are Lot specific be certain that the Lot on the threshold tables matches the Lot of the typing kit e Ifa normalized value for a particular probe falls above the maximum threshold for a negative assignment and below the minimum value for a positive assignment the sample should be considered as indeterminate for this probe e If desired this probe can be removed from consideration or the sample can be typed first assuming the value to be negative and then again assuming the value can be positive e See EXPECTED VALUES section for further information on threshold values LIMITATIONS OF THE PROCEDURE The PCR conditions and assay conditions described require precisely controlled conditions Deviations from these parameters may lead to product failure All instruments must be calibrated according to manufacture s recommendations and operated within manufacture s prescribed parameters 1 Beads must be pre warmed and well suspended prior to use This ensures that the hybridization buffer components are in solution 2 The 56 C incubation requires a high degree of accuracy 0 5 C A thermal cycler should be employed Temperature should be verified within wells of the 96 well thermal cycler plate using a thermocouple e g Bio Rad Model VPT 0300 or equivalent The temperature within wells and among wells should not vary more than 0 5 C
3. 3 Time at 56 C is critical and should not exceed a total of 25 minutes This includes the 20 minute incubation plus no more than 5 minutes to dilute all the samples with Dilution Solution SA PE mixture 4 Once diluted the samples are stable at room temperature for up to 2 hours protect from light Since a full 96 well plate can take up to 1 5 hrs to run through the Luminex Instrument the analysis should be started no more than 30 minutes after dilution to ensure that the last sample is analyzed within the 2 hr limit 5 Do not mix components from other kits and lots Due to the complex nature of KIR typing qualified personnel should review data interpretation and typing assignments Page 5 of 7 LC880RUO 12 05 15 TROUBLESHOOTING Low Bead Count Probe Mix not well suspended Prewarm sonicate and vortex Probe Mix and repeat assay Instrument not Out of Calibration Calibrate Instrument Refer to Luminex IS User s functioning properly manual Sample flow path blocked Remove and sonicate needle Perform backflush Call Immucor Transplant Diagnostics Inc if problem persists 888 329 0255 CON Threshold Sample failed to amplify Low DNA Check DNA concentration and purity Failure or amplified poorly Salts in Master Mix are out of Heat the Master Mix at 37 C for 5 minutes vortex solution gently and spin down briefly Poor Taq Polymerase Use only Recombinant Taq Try Lifecodes 167075 Amplification conditions not within s
4. AND EXPLANATION DNA based KIR typing using PCR amplified DNA is acommon laboratory procedure PCR amplification of DNA is used as the means to enrich for a selected DNA region For KIR typing a subsequent assay is utilized to determine the properties of the amplified DNA Several types of assays such as SSP 1 direct SSOP 2 and reverse SSOP dot blot technologies could be used in KIR typing Like SSOP and reverse dot blot methods LIFECODES KIR SSO Typing kits utilize sequence specific oligonucleotides SSOs to identify which KIR loci are present in a PCR amplified sample It is the set of SSOs employed not the methodologies that determines the ability to distinguish among the various loci present in the PCR amplification Whereas reverse dot blot and SSOP methods employ enzyme labels and colorimetric substrates that require subsequent development the LIFECODES assay is a homogenous multiplex system That is all SSOs are analyzed simultaneously and the entire assay is carried out in a single reaction vessel with the addition of a single reagent PRINCIPLES OF THE PROCEDURE The LIFECODES KIR SSO Typing procedure is based on the hybridization of labeled single stranded PCR product to SSO probes Amplification of DNA using PCR typically employs equimolar amounts of both forward and reverse primer to generate a double stranded DNA product However if the amount of one primer is in excess relative to the other the reaction will generate some single s
5. as potentially infectious Use Universal Precautions when handling 4 Dilution Solution Probe Mix TAQ Polymerase and R Phycoerythrin Conjugated Streptavidin contain hazardous compounds Avoid contact with skin and eyes and dispose of accordingly See Material Safety Data Sheets for additional information C Storage Instructions 1 Refer to kit component packaging label for proper storage temperatures 2 Probe mixes and R Phycoerythrin Conjugated Streptavidin are light sensitive KEEP FROM LIGHT DO NOT FREEZE 3 Do not use components past their expiration date D Purification or Treatment Required for Use See Specimen Collection and Preparation E Instability Indications 1 If salts have precipitated out of solution during shipping or storage resolubilize completely prior to use by vortexing at room temperature 18 30 C 2 Donot use R Phycoerythrin Conjugated Streptavidin that has frozen during shipment or storage INSTRUMENT REQUIREMENTS 1 Luminex Instrument and XY Platform Product Number 888300 31 0 2 Thermal Cycler equipped with a heated lid adjustable ramp times a minimal thermal range of 2 C 100 C and accuracy of at least 0 5 C Conditions for thermal cyclers may need to be altered in order to optimize the profiles A thermal profile may be obtained by contacting Technical Service Tel 203 328 9500 or 888 329 0255 Page 2 of 7 LC880RUO 12 05 15 SPECIMEN COLLECTION AND PREPARATION A DNA can be prepare
6. failure SPECIFIC PERFORMANCE CHARACTERISTICS Specific performance characteristics are to be determined REFERENCES From SUMMARY AND EXPLANATION Section General KIR References 1 Hsu K et al 2002 The Journal of Immunology 169 5118 Vilches C and Parham P 2002 Ann Rev Immunol 20 217 2 Crum KA et al 2000 Tissue Antigens 56 313 Marsh SGE et al 2003 Human Immunology 64 648 LIMITED LICENSE This product is not supplied with Taq polymerase and does not include a license under the foreign patents owned by F Hoffmann La Roche Ltd and Roche Molecular Systems Inc to practice the Polymerase Chain Reaction PCR process described in said patents Further information on the need to obtain purchasing licenses in your country may be obtained by contacting F Hoffmann La Roche Ltd or Roche Molecular Systems Inc The purchase of this product includes a limited non transferable license under U S patent 5 981 180 or its foreign counterparts owned by Luminex Corporation to perform multiplex analysis of clinical specimens for KIR typing Manufacturer Immucor Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 USA Phone 203 328 9500 888 329 0255 Fax 203 328 9599 European Technical Service Phone 32 3 385 4791 This document last revised and issued Rev 12 2015 05 15 Page 6 of 7 LC880RUO 12 05 15 TRADEMARKS USED AB Gene AB Gene House QIAAmp Qiagen Inc Costar Corning Incorporated GelSt
7. warmup Warm Probe Mix in a 55 60 C heat block for at least 5 10 minutes to thoroughly solubilize components in probe mixture Pre warm the thermal cycler to be used for hybridization to 56 C Sonicate briefly 15 sec then vortex Probe Mix for about 15 seconds to thoroughly suspend the beads Aliquot 5 uL of locus specific PCR product into a thermal cycler 96 well plate Costar No 6509 Aliquot 15 uL of Probe Mix into each well When aliquoting probe mix to more than 10 wells gently vortex Probe Mix after each set of ten Seal plate with Microseal film Hybridize samples in the pre warmed thermal cycler at 56 C 56 C HOLD for 20 minutes While the samples are hybridizing prepare a 1 200 Dilution Solution SA PE mixture Combine 170uL Dilution Solution DS and 0 85 uL 1mg mL SA PE per sample Itis recommended to make enough Dilution Solution Mixture for n 1 samples to account for pipetting loss See Table 3 Keep Dilution Solution SA PE mixture in the dark at room temperature SA PE is light sensitive The Dilution Solution may be warmed at 45 C for 5 minutes and vortexed upon arrival to ensure all components are in solution Dilution solution must be at room temperature before making the mixture Prepare prior to use and discard any remaining portion Table 3 Dilution Solution Preparation of Samples PUUA on OOO O Volumes DS 170uL 0 85uL 850uL 4 25uL 1700uL 8 5uL 3400uL 17uL 8500uL 42 5uL Note DO NOT CANCEL
8. HYBRIDIZATION PROGRAM BEFORE REMOVING THE TRAY FROM THE THERMAL CYCLER 8 9 After the hybridization period while the tray is still on the thermal cycler at 56 C dilute each sample with 170uL of the prepared Dilution Solution SA PE mixture Remove the sample tray from the thermal cycler and place in the Luminex Instrument D Analyze sample using the Luminex Instrument For best results assay the samples immediately using the Luminex Instrument Samples can be read up to 30 minutes after being diluted If not read immediately protect samples from light 1 2 Turn on the Luminex Instrument between 30 minutes and 4 hours before assaying the samples Prior to analyzing the samples on the Luminex Instrument set up a Batch Run by which the samples will be analyzed a Select Create a New Batch from the File menu Add Batch for KIR 1 Do the same for KIR 2 The Batch Templates are provided and are named KIR 1 and KIR 2 Please note that the template versions are lot number specific and correspond to the kit lot numbers Page 4 of 7 LC880RUO 12 05 15 Follow the stepwise instructions that appear on the screen for creating batches When naming the batch do not include commas in the name because information after a comma will be lost upon exportation of the data For further instructions on creating batches and multibatches refer to the Luminex IS User s Manual b Click the eject icon to eject the plate holder P
9. IMMUCOR LIFECODES Immucor Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 USA Tel 203 328 9500 or 888 329 0255 Fax 203 328 9599 WWW IMMUCOR COM Product Documentation available at www lmmucor com PRODUCT INSERT LIFECODES KIR SSO TYPING KIT for use with Luminex For Research Use Only Not for use in diagnostic procedures TABLE OF CONTENTS Definition of Symbols cc0ceeeeeees 1 Directions for USe aannnnnnnnnnnnnnnnnnnnnnnns 3 Reagents by Catalog Number 2 A Purify Genomic DNA 0ccc cece eee 3 Summary and Explanation 2 B Amplification ccc cece cee ene ee eee aees 3 Principles of the Procedure 5 2 C HybridiZation ccccceeeeeeeeeeeeeeenes 5 FO AGCINES errian S 2 D Analyze with Luminex Instrument 5 A Identification cccc cece ee eeeee enna 2 FRC SUNES aque ccicuen seem sndivenvinsweseuacaceexmseccmanecnds 5 B Warnings and Cautions 6 2 Limitation of the Procedure 0csssee00 5 C Storage Instructions ee eee 2 TroubleShooting cccceeeeseceeeeeeeneeeeeeenes 6 D Purification or Treatment for Use 2 Expected Value s ccccceeeeeeeeeeeeeeeeeeees 6 E Instability Indications 4 2 Specific Performance Characteristics 6 Instrument Requirements 05 2 REfFErENCES
10. ar Cambrex Bio Science Microseal Bio Rad Laboratories Inc IDNA Agarose Cambrex Bio Science Luminex Luminex Corp APPENDIX A Gel Electrophoresis The PCR reactions performed in the LIFECODES KIR Typing Kits are designed to produce both double and single stranded products which are the predominant products that hybridizes to the SSOs For quality assurance or to trouble shoot an experiment it might be necessary to perform gel electrophoresis to examine the PCR reaction for the presence of amplified DNA Materials Required as listed or equivalent e Electrophoresis Grade Agarose Cambrex IDNA Agarose No GelStar Nucleic Acid Gel Stain Cambrex No 50535 50170 UV Transilluminator ChromatoVUE UVP Inc Model TM36 Electrophoresis apparatus power supply Photographic imaging system 1X Gel Buffer 40xTAE Promega No V4281 The relative migration of the single stranded product is dependent upon the gel concentration and buffer system employed Approximate migrations for each amplification are listed below for samples run in a 2 Agarose gel in 1X TAE buffer Electrophoresis Conditions 1 Remove GelStar Nucleic Acid Stain Cambrex No 50535 from freezer to thaw Keep in dark 2 The gel used for this procedure must be 2 i e for a 200ml gel bed use 4 grams of agarose to 200mL 1X TAE Dilute from 40X TAE Add 10uL GelStar Nucleic Acid Stain to the molten agarose When pouring the gel be sure to leave ample roo
11. ces in amplified DNA and to identify the possible loci in the sample For the LIFECODES KIR Typing procedure probes are attached to LuminexMicrospheres designed for use with the Luminex Instrument Up to 100 different populations of Luminex Microspheres can be mixed together and analyzed by the LuminexInstrument because each population of microspheres can be distinguished by its unique fluorescence signature or color A different SSO probe can be attached to each color microsphere Therefore a mixture of several probes can be distinguished from each other by virtue of their association with particular color microspheres The Luminex Instrument is also able to quantify the relative amounts of labeled PCR product hybridizing to each Luminex Microsphere Therefore the relative signal obtained with the SSO probes in the LIFECODES assay as with other SSOP methods can be used to assign the probes as having positive or negative reactivity with the amplified DNA sample see Results section This in turn provides the information needed to determine the KIR phenotype of the sample REAGENTS A Identification See Reagents By Catalog Number above for complete listing of products and catalog numbers B Warnings or Cautions 1 For Research Use Only Not for use in diagnostic procedures 2 Separate pipettes should be designated for Pre PCR manipulations as well as for Post PCR manipulations 3 Biohazard All biological and blood samples should be treated
12. d using any preferred method such as Ql AAmp Qiagen from whole blood cord blood stain cards and buccal swabs CAUTION All biological and blood samples should be treated as potentially infectious USE UNIVERSAL PRECAUTIONS WHEN HANDLING B DNA extracted from blood preserved in heparin cannot be used in this assay C The isolated DNA should be in 10 mM TRIS pH 8 0 9 0 or in nuclease free water If a chelating agent such as EDTA is present the final concentration of the chelating agent should not exceed 0 5 mM D The presence of alcohol detergents or salts may adversely affect DNA amplification E Final DNA concentration should be 10 50 ng UL F Absorbance measurements of the DNA sample at 260 and 280nm should give a ratio of 1 65 to 2 0 G DNA can be used immediately after isolation or stored at 20 C for up to 1 year Repeated freeze thawing should be avoided since this can result in DNA degradation PROCEDURE A Materials Provided See tables on page 2 for specific information Master Mix MX K1 and MX K2 e Dilution Solution DS Probe Mix BM k e Threshold Table Probe Hit Chart s B Mater als Reagents and Equipment Required but Not Provided as listed or equivalent R Phycoerythrin Conjugated Streptavidin SA PE 1mg mL Lifecodes Cat No 628511 e Bath Sonicator Fisher Scientific No FS15 or FS20 Luminex Sheath Fluid 1x or 20x Lifecodes Cat No 628005 or e Microcentrifuge 628025 l Barrier filter ti
13. ion for n 1 samples using the indicated amount of each component per reaction except for DNA Bring to a final volume of 20uL per reaction with nuclease free water Gently vortex spin down and place on ice Pipette the appropriate amount of Genomic DNA 50ng into the PCR tubes Aliquot the amplification mix into the PCR tubes containing the genomic DNA The total volume of amplification mix and genomic DNA should equal 20uL for each sample reaction Cap tubes tightly to prevent evaporation during PCR Place samples in the thermal cycler and run program See Table 2 Page 3 of 7 LC880RUO 12 05 15 Table 1 Reaction Components for Amplification Component Amount per PCR sample reaction LIFECODES Master Mix MX K1 or MX K2 eu Genomic DNA 10 50ng uL Total of 50ng Taq polymerase 0 2uL 1 0U Nuclease free water To 20uL final volume Table 2 Thermal Cycler Conditions for Amplification Note C H Aaa ode eae oo Step Temperature and Incubation Time of Cycles 1 95 C for 2 min 1 94 C for 30 sec 2 59 C for 90 sec 40 72 C for 30 sec 72 C for 15 min 1 4 4 C forever To be sure of sample amplification refer to Product Gel Electrophoresis Appendix A ybridization Be sure hybridization buffer components of the LIFECODES Probe Mix are solubilized and that the beads are thoroughly suspended Turn on the Luminex Instrument and XY Platform to allow for 30 minute
14. lace the 96 well thermal cycler plate containing the samples in the XYP heater block present on the plate holder c Click the Retract icon The samples are now ready to be analyzed A prime step should be performed before starting the run After the samples have been run through the instrument a sanitization step with 70 Isopropanol or 20 household bleach should be performed followed by two wash steps The instrument can be turned off at this point if it is not going to be used for the remainder of the day 3 After a batch is complete the data is exported as a comma separated values csv file These files are named OUTPUT CSV and saved in a folder with the Batch Name entered into the Luminex IS This data is then available for making typing assignments as described below Refer to Luminex IS User s Manual for instrument operation including daily startup calibration maintenance and shutdown procedures RESULTS Sample typing can be done as follows The generated CSV files can be opened and the data processed with common spreadsheet programs such as Microsoft Excel Lotus 123 Corel Quattro Pro or similar software Analysis is comprised of the following steps 1 Verify that the Number of Events for each SSO in each sample is at least 60 This information is found in the DataType Count section of the CSV file 2 Determine that the values for the Consensus probes for each sample are above their minimum Median Fluorescent Intensit
15. m for DNA to run a significant distance 1 to 2 inches USE CAUTION GelStar is a potential Carcinogen NOTE It is possible to run gels with 20uL of 10mg mL Ethidium Bromide in place of GelStar Nucleic Acid Stain Product band intensity will be less in gels containing Ethidium Bromide than in gels containing GelStar USE CAUTION Ethidium Bromide is a known Carcinogen 3 Keep gelin dark and allow to solidify 4 Load a mixture of 2 5uL of each PCR product and 2 5uL 2X loading buffer with visible dye per sample per amplification Let gel run in the dark at approximately 160 volts for 45 minutes or until sample runs far enough to see separate bands for single and double stranded product bromophenol blue band or other visible marker migrates 1 to 2 inches from wells 5 Photograph using UV Transilluminator accompanied by a GelStar Yellow Photographic Filter Cambrex No 50536 CAUTION Wear protective equipment when handling GelStar Nucleic Acid Stain or Ethidium Bromide and when photographing gel using UV Transilluminator 6 Gel analysis Gel Interpretation Page 7 of 7 LC880RUO 12 05 15
16. pecific parameters Run thermal profile on thermal cycler to verify parameters are within specified parameters Low Median Fluorescent Intensity Value MFI Warm dilution solution at 45 C for 5 minutes before use and vortex Store at room temperature Replace R Phycoerythrin Replace R Phycoerythrin Conjugated Streptavidin Streptavidin Multiple SSO Locus specific Amplification conditions not Run thermal Hi eee on thermal cycler to verity failures or sample amplification within specific parameters parameters are within specified parameters fails to yield a KIR typing result DNA sample contaminated DNA partially degraded Re isolate DNA from blood sample Evaporation during hybridization step If not using an entire plate leave one row empty on each side of samples to be assayed to allow plate to be sealed tightly PCR amplification can be verified by gel electrophoresis See Appendix A EXPECTED VALUES Values can fall into three ranges Negative Positive or Indeterminate An indeterminate value represents a range in which neither positive nor negative values have been observed If asample contains indeterminate values for a particular SSO probe the sample should be re assayed for confirmation It may also be necessary to re isolate DNA from the sample and re amplify and re assay As noted in the Limitations of the Procedure section it is critical to precisely follow the protocol Any deviations can lead to sample typing
17. ps Recombinant Taq Polymerase for kits purchased without Taq e Luminex Calibration beads Cal 1 Cal 2 Con 1 and Con 2 Lifecodes Vortex Mixer Cat Nos 628006 628007 628008 and 628009 respectively Nuclease free water Lifecodes Cat No 757003 20mL e Pipettors Multichannel pipettors and tips 1 20uL 20 200uL 1000uL PCR tubes and caps AB Gene 2ml Thermo Strip No AB0451 G Thermal cycler PCR 96 well plates Costar No 6509 Microseal Film Bio Rad No MSA 5001 or Tape Costar No 6570 Thermal cycler see Instrument Requirements page 2 Spreadsheet software for viewing comma separated value csv files Heat Block Fisher Scientific Standard Heatblock No 13259 030 70 Isopropanol or 20 Bleach DIRECTIONS FOR USE NOTES Probe Mix and SA PE are light sensitive keep away from light and do not freeze Warm beads at 55 60 C for at least 5 10 minutes to thoroughly solubilize components in probe mixture Sonicate briefly 15 sec then vortex probe mix for about 15 seconds to thoroughly suspend the beads Take extreme caution in the aliquoting process using calibrated pipettes Failure to do so may result in reagent loss and sample failure All temperatures must be precisely maintained Fluctuations as little as 0 5 C can affect results At the hybridization stage samples should not remain in the diluted state at 56 C for more than 5 minutes see Results section lt is recommended to assay
18. the amplified samples as soon as possible If the samples can not be run on the Luminex Instrument the same day the amplified product can be stored up to 3 days at 2 8 C prior to use For longer storage store at 20 C up to one week until ready to assay The amplified product can only be frozen and thawed once Repeated freezing and thawing will result in degradation of amplified samples and will yield poor results if assayed lt is recommended that one negative and positive control be run with each test SSO probes that react with loci present in all individuals are used as Consensus Probes Values obtained with the Consensus SSOs from positive controls should exceed the threshold value for the SSO as set forth in the Threshold Table Worksheet Purify genomic DNA using method of choice final concentration should be 10 50 ng uL Adjust if necessary with nuclease free water Keep all samples at similar concentrations DNA amplification PCR Allow the Master Mix for the appropriate loci to warm to Room Temperature 18 30 C Gently vortex the reagents for approximately 10 seconds This will ensure the salts are in solution Spin briefly 5 10 seconds in microcentrfuge to bring contents to the bottom of the tube The multiplex amplification of the KIR loci is divided into two amplification reactions that utilize the KIR 1 Master Mix MX K1 and the KIR 2 Master Mix MX K2 Using Table 1 below prepare the components for amplificat
19. tranded DNA product in addition to double stranded product During the initial cycles of the LIFECODES amplification step double stranded DNA is generated Once the limiting primer is exhausted the remaining primer uses the double stranded product as a template for generation of single stranded DNA This method generates both double stranded and single stranded products that upon denaturation will both participate in the hybridization reaction Each of the different probes may be homologous to a sequence within the amplified DNA that is unique to a locus or group of loci In other words these probes are designed so that each probe preferentially hybridizes to a complementary region that may or may not be present in the amplified DNA In addition the amplified DNA is also hybridized to one or more Consensus probes homologous to loci present in all samples SSO Typing can be affected by the type of biological material method of purification amount and integrity of genomic DNA Therefore the signal obtained for the Cons ensus probe s can serve as an indicator of the success of the amplification and hybridization procedures Also the signal obtained with the Consensus probe can be used to normalize the signal of the locus specific probes and correct for variations in the amount of amplified product in the hybridization reaction The analysis of the results generated from the SSO typing can be used to determine the presence or absence of particular DNA sequen
20. y or MFI The minimum thresholds are lot specific and can be found in the Threshold Table Caution e To obtain reliable results there must be sufficient data gathered by the Luminex Instrument e Collect at least 60 events for each SSO e The LIFECODES Probe Mix contains two SSOs designated CON100 and CON200 that hybridize to KIR locus 3DP1and 3DL3 respectively These act as internal controls to verify that the PCR reactions and hybridizations worked e Ifthe minimum value is not obtained for these SSOs the sample may not produce the correct typing and the sample test should be repeated 3 Subtract the Background Control value for each probe from the sample values producing the background corrected data set Background Control values are found in the Threshold Table and are lot specific Background values are average MFI values for each bead to compensate for background noise due to bead variation 4 For each sample divide the background corrected data for each probe by the background corrected value for the corresponding consensus probe producing the normalized data set MEI Probe MFI Control blank for probe MFI Consensus MFI Control blank for consensus For each probe record the normalized value on the Threshold Table Worksheet 6 Once all values have been assigned the probe hit pattern i e the combination of all positive and negative assignments for a given sample can be compared with the Probe Hit Table provided
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