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Sample & Assay Technologies ipsogen® JAK2

1. o 00000011111111111111 1 080887 6 Figure 11 Example of results shown in Note The file contains both raw data and standardized data Only standardized data must be considered Excel file These data are given in the Quantitative analysis of channel Cycling A Green and Quantitative analysis of channel Cycling A Yellow sections of the table The data intended for interpretation are those acquired at PCR cycle 50 in circles on the right 20 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Protocol qPCR on Applied Biosystems and ABI PRISM instruments Using 96 wells plate qPCR equipment we recommend performing all measurements in duplicate as indicated in Table 5 Table 5 Number of reactions for Applied Biosystems 7300 and 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments Samples Reactions JAK2 V617F primers and probes mix PPM VF 56 reactions 19 DNA samples 19 x 2 reactions 2 x 2 reactions MP VF MN VF each one tested in duplicate 6 x 2 reactions M1 to M6 each one tested in duplicate 2 DNA controls Reference scale Water control 2 reactions Sample processing on Applied Biosystems 7300 and 7500 ABI PRISM 7000 and 7700 or ABI PRISM 7900HT instruments Figure 12 Suggested plate setup for an experiment with the ipsogen JAK2 MutaScreen RS Kit MP positive control MN negative control M1 to M6 reference scale S DNA sample H2O water control
2. W Poor positioning of a water control higher than MN for FAM measurement or higher than MP for VIC may indicate contamination Note Positioning of the controls may be different on analysis of LightCycler 2 0 instrument data see Figure 33 The water controls should still be located at the bottom left MN E H2O oM 2 M2 5 M3 12 Rn fluorescence FAM 530 nm AMA 31 eM5 50 H2O AM6 78 Rn fluorescence VIC 560 nm Figure 33 Scatter plot of a representative allelic discrimination experiment Instrument LightCycler 2 0 Calculation of normalized FAM VIC ratio and genotyping Calculate the FAM VIC ratios for all the samples Calculate the FAM VIC ratios for the positive control MP the cut off sample M1 the negative control MN and the reterence scale M2 to M6 The ratios must be consistent between duplicates Calculate the average ratio of all duplicates Calculate the normalized ratio NRatio for the cut off sample M1 and for all the samples RatiOsample N RatiOsample Ratio MN Note The gray zone GZ of a test is defined as an area of values where the discriminatory performance is insufficiently accurate A value in the gray zone indicates that the target marker cannot be scored as either present or absent The gray zone must be calculated for each experiment 42 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Calculate the gray zone or the incertitude area around the normalize
3. approximately 10 seconds 9 Load the samples in the thermal cycler according to the manufacturer s recommendations 10 Program the thermal cycler Figure 28 with the program as indicated in Table 13 For programming details of the LightCycler 2 0 instrument refer to the instrument user guide For a better overview the software settings are framed in bold black Note Make sure that the setting is for Quantification and single acquisition of FAM fluorescence and single acquisition of VIC fluorescence in both the amplification cycling step and the final hold at 60 C 2c Light pidas Sara Honld 4 31 21k Haw Capertee nt mu rx E ru mw Ta Jod WER PH T m C grim daas s Rum r4 mel z al Er Targa ma Pass LI Tieg ae di Tiag EO dd seer TESTI 38 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Table 13 Temperature profile for LightCycler 2 0 instrument Temperature 55 C Time 2 minutes Ramp 20 Temperature 95 C Time 10 minutes Ramp 20 Cycling 50 times 92 C tor 15 seconds ramp 20 60 C for 1 minute ramp 20 60 C for 1 minute ramp 20 End point analysis procedure for LightCycler 2 0 instrument 11 At the end of the amplification run click the tab for Online Data Display Figure 29 Open the display menu on the top left of the Current Fluorescence window then write 51 in Acquisition no Cet aren eae l z SEESEN Hj j um i aa Tempesrmbere Wisra
4. ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 21 qPCR on Applied Biosystems 7300 and 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments Note Perform all steps on ice Procedure 1 Thaw all necessary components and place them on ice Components should be taken out of the freezer approximately 10 minutes before starting the procedure 2 Vortex and briefly centrifuge all the tubes approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 3 Prepare the following qPCR mix according to the number of samples being processed All concentrations are for the final volume of the reaction Table 6 describes the pipetting scheme for the preparation of one reagent mix calculated to achieve a final reaction volume of 25 ul A pre mix can be prepared according to the number of reactions using the same primer and probe mix Extra volumes are included to compensate for pipetting error On the Applied Biosystems 7300 and 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments the ipsogen JAK2 MutaScreen RS Kit can be used for analysis of 19 samples in duplicate in one experiment Figure 12 Table 6 Preparation of qPCR mix Number of reactions pl Final Component 56 1 concentration TaqMan Universal PCR Master Mix 2x d n Primers and probes mix 10x Nuclease free PCR grade water 142 5 Ix 285 Sample to be added at step 4 gt eed Total v
5. manual in combination with a validated instrument mentioned in Materials Required but Not Provided page 9 Any diagnostic results that are generated must be interpreted in conjunction with other clinical or laboratory findings It is the user s responsibility to validate system performance for any procedures used in their laboratory which are not covered by the QIAGEN performance studies Attention should be paid to expiration dates printed on the box and labels of all components Do not use expired components Performance Characteristics Nonclinical studies Nonclinical studies were conducted to establish the analytical performance of the ipsogen JAK2 MutaScreen Kit Precision Three dilution levels of genomic DNA from cell lines harboring the JAK2 V617F mutation in wild type DNA were tested The dilutions corresponded to mutation loads of 196 296 and 396 Independent dilution batches were obtained for each level and replicates of these dilutions were tested in 3 independent experiments Ratios obtained for each DNA sample Ratiosampie were compared with the negative control ratio JAK2 100 wild type DNA Ratio c Results are summarized in Table 17 Table 17 Precision data for nonclinical studies Mutation level RatiOsampie gt Rationc CV ratio 1 V617F DNA 10096 n 183 6 8 296 V617F DNA 100 n 72 4 5 396 V617F DNA 10096 n 135 5 1 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 47 Interlaborat
6. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera essential thrombocythemia and myeloid metaplasia with myelofibrosis Cancer Cell 7 387 4 Kralovics R et al 2005 A gain of function mutation of JAK2 in myeloproliferative disorders N Engl J Med 352 1779 5 Baxter E J et al 2005 Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders Lancet 36 1054 6 Tefferi A et al 2009 Myeloproliferative neoplasms contemporary diagnosis using histology and genetics Nat Rev Clin Oncol 6 627 7 Prchal J F and Axelrad A A 1974 Bone marrow responses in polycythemia vera N Engl J Med 290 1382 8 Tefferi A and Vardiman J W 2008 Classification and diagnosis of myeloproliferative neoplasms the 2008 World Health Organization criteria and point of care diagnostic algorithms Leukemia 22 14 9 Barosi C et al 2009 Response criteria for essential thrombocythemia and polycythemia vera result of a European LeukemiaNet consensus conference Blood 113 4829 10 Pardanani A et al 2011 Safety and efficacy of TG101348 a selective JAK2 inhibitor in myelofibrosis J Clin Oncol 29 789 11 Lippert E et al 2006 The JAK2 V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera Blood 108 1865 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 53 Symbols The following symbols may appea
7. Manual Call C VIC Unknown 269 500 JAK2 VIC JAK2 FAM 2 82 1 892 Undetermined 100 00 Manual Call C VIC Unknown 211 520 JAK2 VIC JAK2 FAM 1 249 6 14 Undetermined 100 00 Manual Call C VIC Unknown 270 523 JAK2 VIC JAK2 FAM 1 346 5 894 Undetermined 100 00 Manual Call C VIC Unknown 355 112 JAK2 VIC JAK2 FAM 1 265 6 526 Undetermined 100 00 Manual Call ER VIC Unknown 372 150 JAK2 VIC JAK2 FAM 2 214 2 03 Undetermined 100 00 Manual Call ER VIC Unknown 404 145 JAK2 VIC JAK2 FAM 2 419 2 255 Undetermined 100 00 Manual Call ER VIC Unknown 410 977 JAK2 VIC JAK2 FAM 2 581 2 52 Undetermined 100 00 Manual Call H20 VIC Unknown 395 431 JAK2 VIC JAK2 FAM 0 655 1 345 Undetermined 100 00 Manual Call H20 VIC Unknown 415 223 JAK2 VIC JAK2 FAM D 727 1 241 Undetermined 100 00 Manual Call H20 VIC Unknown 366 685 JAK2 VIC JAK2 FAM 0 606 1 272 Undetermined 100 00 Manual Call Figure 19 Example of results shown in an Excel file 28 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Protocol qPCR on the LightCycler 480 instrument Using 96 well plate qPCR equipment we recommend performing all measurements in duplicate as indicated in Table 8 Table 8 Number of reactions for the LightCycler 480 instrument Samples Reactions JAK2 V617F primers and probes mix PPM VF 56 reactions 19 DNA samples 19 x 2 reactions 2 x 2 reactions MP VF MN VF each one tested in duplicate 6 x 2 reactions M1 to M6 each one tested in duplicate 2 DNA controls Reference sca
8. Pattive Reference OM F Marker Were Detector Detector 1 AK BL Alele B Allele A New Detector New Marker Figure 17 Selecting markers 21 Click Next gt 22 In the Setup Sample Plate dialog box click and drag to select the marker for wells that contain samples Click Finish 23 Select the Instrument tab and change the sample volume to 25 ul 24 Select File Save and then click Save to retain the name you assigned when you created the plate 25 Load the reaction plate into the instrument according to the manufacturer s recommendations Do 26 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 26 Start the post read run Click Post Read The instrument will perform a run of 1 cycle for 60 seconds at 60 C During this run the instrument collects FAM and VIC fluorescence in each well Figure 18 E File View Tools Imstrurent Analyse Window Help 7 Setup instrument Results Vo Instrument Comro Ma Estimated Time Remaining hhimimy sample Hast Sink FostRead m Caeli _ Disconnect Status Stage Hep Time mimssy Sep atate Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Au BISSUOLIETUN mage BHEE Help SUSE e Pau ui pitis Settings sample Volume HL Run Made Standard 7500 gt Data Collection Stage 1 St
9. This product is sold under a licensing arrangement with Epoch Biosciences for use only in in vitro diagnostics and may not be used for any other research commercial clinical research or other use outside of the in vitro diagnostics field JAK2 V617F mutation and uses thereof are protected by patent rights including European patent EP1692281 US patents 7 429 456 and 7 781 199 US patent applications US20090162849 and US20120066776 and foreign counterparts The purchase of this product does not convey any right for its use for clinical trials for JAK2 V617F targeted drugs QIAGEN develops specific license programs for such uses Please contact our legal department at jak2licenses giagen com Trademarks QIAGEN QlAamp HRM ipsogen Rotor Gene QIAGEN Group ABI PRISM Applied Biosystems FAM VIC Life Technologies ARMS AstraZeneca Ltd Excel Microsoft Corporation iCycler Bio Rad Laboratories Inc LightCycler TaqMan Roche Group MGB Epoch Biosciences Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the ipsogen JAK2 MutaScreen RS Kit to the following terms 1 The ipsogenJAK2 MutaScreen RS Kit may be used solely in accordance with the ipsogen JAK2 MutaScreen RS Kit Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with
10. each QIAGEN kit and kit component Discard sample and assay waste according to your local safety regulations 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 General precautions qPCR tests require good laboratory practices including equipment maintenance that are dedicated to molecular biology and compliant with applicable regulations and relevant standards This kit is intended for in vitro diagnostic use Reagents and instructions supplied in this kit have been validated for optimal performance Further dilution of the reagents or alteration of incubation times and temperatures may result in erroneous or discordant data PPM VF reagent may be altered if exposed to light All reagents are formulated specifically for use with this test For optimal performance of the test no substitutions should be made Use extreme caution to prevent DNase contamination which might cause degradation of the template DNA E DNA or PCR carryover contamination resulting in false positive signal We therefore recommend the following E Use nuclease free labware e g pipets pipet tips reaction vials and wear gloves when performing the assay BE Use fresh aerosol resistant pipet tips for all pipetting steps to avoid cross contamination of the samples and reagents E Prepare pre PCR master mix with ded
11. one tested in duplicate 6 x 2 reactions M1 to M6 each one tested in duplicate 2 DNA controls Reference scale Water control 2 reactions 12 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Sample processing on Rotor Gene Q instruments with 72 tube rotor g 97 98 38 sig 0000 0009 i 5n OO O Q Q MA OOOO00 as M6 M Figure 2 Suggested rotor setup for an experiment with the ipsogen JAK2 MutaScreen RS Kit MP VF positive control MN VF negative control M1 to M6 reference scale S DNA sample H O water control Note Take care to always place a sample to be tested in position 1 of the rotor Otherwise during the calibration step the instrument will not perform calibration and incorrect fluorescence data will be acquired Fill all other positions with empty tubes qPCR on Rotor Gene Q instruments with 72 tube rotor Note Perform all steps on ice Procedure 1 Thaw all necessary components and place them on ice Components should be taken out of the freezer approximately 10 minutes before starting the procedure ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 13 2 Vortex and briefly centrifuge all the tubes approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 3 Prepare the following qPCR mix according to the number of samples being processed All concentrations are for the final volume of the reaction Table 3 describes the pipetting scheme
12. probe is cleaved by the 5 3 exonuclease activity of Tag polymerase separating the Reporter dye from the Quencher and thus releasing detectable fluorescence The probe not perfectly matched will be displaced rather than cleaved by the Taq polymerase and no reporter dye is released The fluorescence signal VIC or FAM generated is collected at the end of the PCR end point and immediately indicates the presence of the targeted sequence s in the sample wild type allele mutated allele or both without the requirement of long and laborious post PCR steps which also increase the contamination risk The actual quantity of target sequence is not determined The ipsogen JAK2 MutaScreen RS Kit uses this technology as illustrated see Figure 1 6 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 x d Probe degradation Probe 1 ii DI aa Pees o PEE PEE of FAM signal Probe 2 An Probe degradation N II ee ee zu ern D SUP stantial increase of VIC signal C displacement robe 1 gag ee n Allele 2 Mismatch EN lE No signal Probe displacement Probe 2 JE E ED s K ER Figure 1 TaqMan probe multiplex assay The ipsogen JAK2 MutaScreen RS Kit uses this technology for allelic discrimination ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 7 Materials Provided Kit contents ipsogen JAK2 MutaScreen RS Kit 19 Catalog no 673123 Number of reactions 19 V617F Positive Control MP VF 30 ul V617F
13. the reaction Repeat the PCR run b Inappropriate storage of Store the ipsogen JAK2 MutaScreen RS Kit at kit components 30 to 15 C and keep primers and probes mix PPM protected from light See Reagent Storage and Handling page 11 Avoid repeated freezing and thawing Aliquot reagents for storage Negative controls are positive Cross contamination Replace all critical reagents Repeat the experiment with new aliquots of all reagents Always handle samples kit components and consumables in accordance with commonly accepted practices to prevent carry over contamination No signal even in positive controls a Pipetting error or omitted Check pipetting scheme and the setup of the reagents reaction Repeat the PCR run b Inhibitory effects of the Repeat the DNA preparation sample material caused by insufficient purification ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 45 Comments and suggestions c LightCycler Incorrect Set Channel Setting to F1 F2 or detection channel chosen 530 nm 640 nm d LightCycler No data Check the cycle programs acquisition programmed Select acquisition mode single at the end of each annealing segment of the PCR program Absent or low signal in samples but positive controls okay Poor DNA quality or low Always check the DNA quality and concentration concentration before starting LightCycler Fluorescence intensity too low a Inappropriate storage of
14. 17 18 19 2 In the New Detector dialog box type Allele A in the Name field Figure 15 Leave the Reporter Dye set to FAM Click the Color button select a color and then click OK Figure 15 Click Create Another Figure 15 Hew Detector Nan Bollele A Description Reporter Dye B Quencher Die Color Hotes Cancel Figure 15 Creating detectors In the next New Detector dialog box type Allele B in the Name field Select VIC in the Reporter Dye field Click the Color button select a color and then click OK Click New Marker in the Select Markers dialog box see Figure 14 In the New Marker dialog box type JAK2 in the New Marker Name field Figure 16 Select the Allele A and Allele B detectors as created in steps 16 and 17 or already defined and click OK Figure 16 Mew Marker Hea Marker Mame f JARS Dalag bao chleEeck Lre for bus markar Alele B NK none Allee 5 F Aut nona Cancel Figure 16 Creating markers ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 25 20 In the Select Markers dialog box select JAK2 as created above or a suitable predefined marker and then click Add Figure 17 Note To remove a marker select it and then click Remove Hew Document Wizard Select Markers Select the makers you vell be using in Ihe document Fund a 7
15. 2 percentage agreement ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 49 Table 21 Comparison between methods ipsogen JAK2 MutaScreen Kit and sequencing Results of direct sequencing JAK2 V617F JAK2 V617F gt 2 lt 2 Total JAK2 V617F Mutation 26 27 Results of detected ipsogen JAK2 Inconclusive 0 MutaScreen result testing JAK2 WT method No mutation 2 detected Total 28 Table 22 Comparison between methods ipsogen JAK2 MutaScreen Kit and sequencing Agreement 96 95 CI 906 Positive data Agreement between ipsogen JAK2 92 9 4 98 0 MutaScreen Kit and sequencing Negative data Agreement between ipsogen JAK2 3 99 2 MutaScreen Kit and sequencing Total agreement 83 5 97 9 Contidence intervals were calculated according to CLSI EP12 A User Protocol for Evaluation of Qualitative Test Performance Approved Guideline Multicenter study on 228 patient samples DNA samples from patients were analyzed with home brew techniques in 13 laboratories contributing to an interlaboratory study In each laboratory 3 experiments were performed using DNA from cell lines as described for the nonclinical precision data see above and with DNA from 10 patients available in the laboratory 50 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 The 228 samples with a known JAK2 genotype were tested in parallel with the ipsogen JAK2 MutaScreen Kit and by home brew methods including qualitative PC
16. 6 000 44 000 42 000 40 000 38 000 36 000 34 000 32 000 t 30 000 3 26 000 26 000 24 000 3 22 000 20 000 18 000 16 000 14 000 12 000 10 000 8 000 6 000 4 000 2 000 5 000 10 000 15 000 20 000 25 000 30 000 35 000 40 000 45 000 0 000 Fluorescence 523 568 al ee Figure 34 Analysis of healthy donors LightCycler 480 analysis of 38 healthy donors 9 with the ipsogen JAK2 MutaScreen RS Kit cat no 673123 Positive results in duplicate 4 correspond to a reference scale supplied with the kit VIC fluorescence values are plotted on the x axis and FAM values are plotted on the y axis 52 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor Cited references 1 Ma W et al 2009 Mutation profile of JAK2 transcripts in patients with chronic myeloproliferative neoplasias J Mol Diagn 11 49 2 James C et al 2005 A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera Nature 434 1144 3 Levine R L et al 2005
17. 72 N 50 12 05 642E 03 516E 03 389E 03 262E 03 amp 4 HHH H H oH H HH HH H H HEHEHE HEHEHE HH O09 8400000000 0 01995 om 54 O S L s3 ID MSPPOI 1 2 3 456789391848 RER HH HH HR HR HR HH HH RR 8 8 HH HR RR RR 8 HR UR RU 5 1 pC 3 70E 03 3 09E 03 2488 03 187E 02 amp 4 amp W amp H S HW HK HH 8 HH HH HH HR HH 8 OR R0 8 R88 HH HH 00000 01275 O 55 2 pC 419E 04 7 84E 04 115E 03 SEHR HH HH HH HH HH HH HR OR HR HH HH HH HH 8880000070 012198 5 3 nC 220E 03 244E 03 2698 03 2939 03 4 amp amp W amp Od amp HW HH HR HR HR HR HR HR HR HR OO OO OO TI TAT TAT TAT TAT 1 07952 Oo s 4 nC 2646 03 3 09E 03 354E 03 3 99E 03 amp amp HHH HH M HW d 11111111 082 ss 5 S 597E 03 5 28E 03 460E 03 3 92E 03 4 amp M HHH HH MH d 11111111 08305 2 6 S 6 70E 03 578E 03 487E 03 3 95E 03 amp amp HHH HHH V 4 1 CO U m n 1111111 1 0 7848 O amp o 7 h20 733E 04 102E 03 130E 03 159E 03 amp 4 4 S HHH HH HU 4 8 149E 8 h20 227E 04 6 86E 04 114E 03 160E 03 4 4 HHH HHH HHH HF FF RF FR OR OF FR RR RR HO OH OH OH OH 356E 0 e 3 0 117E 02 101 02 847E 03 6 83E 03 amp amp 4 amp amp W amp KW K KW WR HORE 800002020 1111111 111111111 1 078928 U 3 oo 0 5 76E 03 4916 03 406E 03 322E 020 amp amp 4 M W W amp HU KHU HH HR 84400002020 1111111 1111111 1 1 1 080408 ZE 1 05 516E 03 459E 03 402E 03 3 45E 03 amp 00000111111111111111111 1 078975 es 2 05 519E 03 480E 03 4 42E 03 404E 02 amp 4
18. 77777777777 7 71364 72198062 O 9 0 5 76232 5 84288 589174 5 90149 666666 77788888339232329998 amp HH amp 10405 10602097 1 0 0 5 81827 5 91948 5 92752 5 92732 6 6 6 6 6 6 77777888888332392398 84 9 amp 10 2008 10 425509 oO wo 1 05 5 79919 5 82568 5 85641 584186 6 6 6 6 6 6 i 77788888332323239u9 4 4 HHH 105793 10 749218 20 2 05 6 13053 6 16999 6 16846 620520 6 6 6 6 6 6 7777888832392399 4 4 4 1l 44 125759 12972075 21 gt z Channel Cycling A Yellow 23 ID MSPPOI 1 2 3 45678989 DEE IE IE IE IE IE IE IE IE RHEE 49 50 O 24 ii pc 459685 462247 46819 4 634715 5 5 5 5 5 16666666777777777798 8 8 7 93092 8 0828056 25 2 pC 466362 472438 4718198 474741 5 5 5 5 5 5 5566 6666677777778888 8 8 06134 8235294 CY 26 3l nC 453005 467231 464528 347200215 55555 X IHRER HR RR 8 08 8 8 4 8 HHH 12335 42626596 2 4 nc 5 02795 5 05531 5 22326 5 225077 555555 PAR OR OH OH HORROR R8 8 HHH HHH HH 531528 52465224 2 5 S 5 02092 5 01981 5 04675 51214 5 5 5 5 5 5 5 eee O8 OR OR OR OR HHH HH HH HH 475771 47 706823 2 6 S 4 71329 471646 476223 471725 55 5 55 T LII OS OH OR OR OH HH HH 8 8 8 HHH HHH 42 067 42237559 30 7 h20 487286 493455 492549 5008725 55 55 55 5 5 5 5 5 51575 5 5 5 5 5 6 61616 6 66 6 6666666666666666 6 14522 6020979 8 h20 456801 4588601 46986 472088 55 5555 555555555555555555555555666656666666 6 6 584573 58762493 9 0 462588 460961 471013 466348555555555555555556666679 1 amp amp amp W amp HH amp HHH H
19. 9 08 1 Sample 1 610 1 822 734 1 Sample 1 560 1 Sample 1 530 2 30182 2 Sample 2 610 2 8 4428 2 Sample 2 560 2 Sample 2 530 3 28495 3 Sample 3 610 3 Sample 3 560 3 Sample 3 530 4 29526 4 Sample 4 610 4 8 2887 4 Sample 4 560 4 Sample 4 530 5 29450 5 Sample 5 610 5 6 2669 5 Sample 5 560 5 Sample 5 530 b 29958 6 Sample 6 610 6 5 4154 6 Sample 6 560 6 Sample 6 530 7 30045 7 Sample 610 7 64520 7 Sample 7 560 7 Sample 7 530 B 32822 Sample 5 610 6891936 6 Sample 8 560 B Sample 6 530 930274 98 Sample 9 610 96 5557 9 Sample 9 560 9 Sample 9 530 10 28335 10 Sample 10 610 10 7 9713 10 Sample 10 560 10 sample 10 530 11 2 8275 11 Sample 11 610 11 7 9774 11 Sample 11 660 11 gt sample 11 530 12 28351 12 Sample 12 610 12 8 0171 12 Sample 12 660 12 gt Sample 12 530 13 28511 13 Sample 13 610 13 8 3725 13 Sample 13 550 13 sample 13 530 14 283b 14 Sample 14 610 14 8 0217 14 Sample 14 560 14 sample 14 530 15 288308 15 Sample 15 610 15 8 4337 15 Sample 15 6560 15 sample 15 530 16 28885 16 Sample 16 610 16 8 1498 15 Sample 16 560 15 sample 16 530 1 30152 17 Sample 17 610 17 8 4901 17 Sample 17 560 17 gt sample 17 530 Figure 31 Example of LightCycler 2 0 results shown in an Excel file 40 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Interpretation of Results Obtain a file suitable to extract export
20. HH HH HHH 44 9936 45977775 3 10 0 4 7134 4 87286 482816 480475555555555555555555666 43 4785 44 035357 3 05 46817 472921 471081 472123 5555555555555555555667 42478 42 437756 35 2 05 48555 50297 5 04229 504835555555555555555666666 P R rou n 5 47 002 47 084982 36 3 O 3 lU MSPPUI 1 2 3 5b5 0989g9IUS9 4 HH RR RR OR RR RR UR HH HH HH RR OR RR RR OR HHH 50 39 1 pC 524E 03 507E 03 4 89E 03 472E 02 amp 00000111111111111111111 1 087425 0 87453 40 2 pc 65E 03 2986 03 31E 03 3 34E 03 amp amp 4 4 W W W KH WR RE HERE 29000020201 111711711717111111 1 09943J 0 9446 O a 3 nC 41E 03 223E 03 205E 03 199E 03 amp amp 4 amp W W H M HW HK HH HH HR V KH EEE EEE HOOK 9200002020 0194 0 13137 az 4 nC SS SRSA Rew ee ated SON TEE SPEC MT EEE een anew er vhs sor 9404781101 0 0 DIDI GINE DIOENA Oo m 5 s S 04 100000000 0248 025652 44 6 S am e name LIE IE JE JE FAM CO umn 100000000 02293 0 23531 es 7 mo p DA OR 577E 0P 5 81E 03 Oo a Umso m pn rms ESE DA 8 8 4 224E 0 2 47E 03 a 9 0 6 49E 03 5 73E 03 438E 03 422E 03 4 amp amp HW HW S H HH HH H H H H H HH OH OH HR HH OH OF HR HH RR 008980892 80002020 DM 0 12178 Q a 0 0 3 79E 03 3 24E 03 270E 03 216E 03 amp amp amp 4 amp H W M H W KH H H KH W KO W amp WW VOR HR 8 HH HH 88848000000 01994 0 13259 4s 1 05 4156 03 347E 03 280E 03 212E 03 amp amp 4 4 W amp W amp W W amp HW KW OH HH HH 8 HH HH HR HH HH HH 0000000000 01566Y 0 159
21. January 2013 ipsogen JAK2 MutaScreen RS Kit Handbook Wig Version Quantitative in vitro diagnostics For use with Rotor Gene Q Applied Biosystems ABI PRISM and LightCycler instruments Ce 673123 uel QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden GERMANY R1 a 107251 3EN QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Intended Use 4 Summary and Explanation 4 Principle of the Procedure 6 Materials Provided 8 Kit contents 8 Materials Required but Not Provided 9 Warnings and Precautions 10 General precautions 10 Reagent Storage and Handling 11 Procedure 12 Sample DNA preparation 12 Storing nucleic acids 12 Protocols E GPCR on Rotor Gene Q instruments with 72 tube rotor 12 BI gPCR on Applied Biosystems and ABI PRISM instruments 21 E GPCR on the LightCycler 480 instrument 29 E GPCR on LightCycler 2 0 instrument 36 Interp
22. MutaScreen RS Kit Handbook 01 2013 Table 1 WHO criteria for the diagnosis of MPN adapted from reference 8 Criteria for a diagnosis of polycythemia vera PV Major 1 Hemoglobin Hgb gt 18 5 g dl men or gt 16 5 g dl women or Hgb or hematocrit Hct gt 99th percentile of reference range for age sex or altitude of residence or Hgb gt 17 g dl men or gt 15 g dl women if associated with sustained increase of 22 g dl from baseline that cannot be attributed to correction of iron deficiency or Elevated red cell mass gt 25 above mean normal predicted value Minor 1 Bone marrow trilineage myeloproliferation 2 Subnormal serum erythropoietin level 3 Endogenous erythroid colony EEC growth Criteria for a diagnosis of essential thrombocythemia ET Major 1 Platelet count 2450 x 10 I 2 Megakaryocyte proliferation with large and mature morphology No or little granulocyte or erythroid proliferation 3 Not meeting WHO criteria for chronic myeloid leukemia CML PV primary myelofibrosis PMF myelodysplastic syndrome MDS or other myeloid neoplasm No evidence of reactive thrombocytosis Minor Criteria for a diagnosis of primary myelofibrosis PMF Major 1 Megakaryocyte proliferation and atypia accompanied by either reticulin and or collagen fibrosis or In the absence of reticulin fibrosis the megakaryocyte changes must be accompanied by increased marrow cellularity granulocytic proliferation and often decre
23. Negative Control MN VF 30 ul Reference Scale M1 M1 VF 30 ul Reference Scale M2 M2 VF 30 ul Reference Scale M3 M3 VF 30 ul Reference Scale M4 MA VF 30 ul Reference Scale M5 M5 VF 30 ul Reference Scale M t M6 VF 30 ul Primers and probes mix JAK2 V617F ipsogen JAK2 MutaScreen RS Kit Handbook English PPM VF 10x 145 ul Positive control 100 V617F DNA t Negative control 100 wild type DNA 0 V617F DNA Reference scale genomic DNA dilutions 3 Mix of specific reverse and forward primers for the JAK2 gene specific V617F FAM probe and wild type VIC probe Note Briefly centrifuge tubes before use Note Analyzing unknown samples with the ipsogen JAK2 MutaScreen RS Kit requires the extraction of genomic DNA Reagents needed to perform DNA extraction e g QIAGEN QlAamp DNA Mini Kit cat no 51304 are not provided and must be validated in combination with the kit 8 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Materials Required but Not Provided When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier Reagents E Nuclease tree PCR grade water E Nuclease tree 1x TE buffer pH 8 0 e g Life Technologies cat no 12090 015 Butter and Tag DNA polymerase The validated reagents are TaqMan Universal PCR Master Mix Master Mix PCR 2x Lite T
24. R allele specific PCR fluorescence energy resonance transfer FRET sequencing allele specific oligonucleotide PCR RFLP and allelic discrimination Results of the comparisons are shown in Table 23 2 x 3 contingency table and Table 24 percentage agreement Table 23 Comparison between methods ipsogen JAK2 MutaScreen Kit and home brew methods Results of home brew testing JAK2 V617F JAK2 V617F gt 2 lt 2 Total JAK2 V617F Results of Mutation 139 142 ipsogen detected JAK2 Inconclusive 5 MutaScreen result testing JAK2 WT method No mutation 3 64 detected Total 147 22 Table 24 Comparison between methods JAK2 MutaScreen Kit and home brew methods Agreement 96 95 CI 96 Positive data Agreement between ipsogen JAK2 97 9 94 0 99 3 MutaScreen Kit and home brew Negative data Agreement between ipsogen JAK2 87 1 98 4 MutaScreen Kit and home brew Total agreement 93 8 98 7 Confidence intervals were calculated according to CLSI EP12 A User Protocol for Evaluation of Qualitative Test Performance Approved Guideline ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 51 Robustness testing of samples from healthy donors DNA samples from103 healthy blood donors were analyzed with the ipsogen JAK2 MutaScreen RS Kit All of the samples were detected as JAK2 wild type Analysis of 38 samples with the LightCycler 480 instrument is shown in Figure 34 Fluorescence j Scatter Plot 50 000 48 000 4
25. Store the ipsogen JAK2 MutaScreen RS Kit at kit components 30 to 15 C and keep primers and probes mix PPM protected from light See Reagent Storage and Handling page 11 Avoid repeated freezing and thawing Aliquot reagents for storage b Very low initial amount of Increase the amount of sample DNA target DNA Note Depending of the chosen method of DNA preparation inhibitory effects may occur LightCycler Fluorescence intensity varies a Pipetting error Variability caused by so called pipetting error can be reduced by analyzing data in the F1 F2 or 530 nm 640 nm mode b Insufficient centrifugation The prepared PCR mix may still be in the of the capillaries upper vessel of the capillary or an air bubble could be trapped in the capillary tip Always centrifuge capillaries loaded with the reaction mix as described in the specific operating manual of the apparatus c Outer surface of the Always wear gloves when handling the capillary tip dirty capillaries 46 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Quality Control Quality control of the complete kit has been performed on a LightCycler 480 This kit is manufactured according to ISO 13485 2003 standard Certificates of Analysis are available upon request at www giagen com support Limitations The users must be trained and familiar with this technology prior to the use of this device This kit should be used following the instructions given in this
26. a vera PV 50 60 of patients with essential thrombocythemia ET and in 50 of patients with primary myelofibrosis PMF JAK2 V617F has been also detected in some rare cases of chronic myelomonocytic leukemia myelodysplasic syndrome systemic mastocytosis and chronic neutrophilic leukemia but in 096 of CML 6 The mutation corresponds to a single nucleotide change of JAK2 nucleotide 1849 in exon 14 resulting in a unique valine V to phenylalanine F substitution at position 617 of the protein JH2 domain It leads to constitutive activation of JAK2 hematopoietic transformation in vitro and erythropoietin independent erythroid colony EEC growth in all patients with PV and a large proportion of ET and PMF patients 7 JAK2 V617F represents a key driver in the transformation of hematopoietic cells in MPN but the exact pathological mechanisms leading with the same unique mutation to such different clinical and biological entities remain to be fully elucidated Traditionally the diagnosis of MPNs was based on clinical bone marrow histology and cytogenetic criteria The discovery of a disease specific molecular marker resulted in both simplification of the process and increased diagnostic accuracy Detection of the JAK2 V617F mutation is now part of the reference WHO 2008 criteria for the diagnosis of BCR ABL negative MPN Table 1 and presence of this mutation is a major criterion for diagnostic confirmation 4 ipsogen JAK2
27. and a qPCR assay based on the amplification refractory mutation system ARMS principle 5 Results of the comparison are shown in Table 19 2 x 3 contingency table and Table 20 percentage agreement 48 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Table 19 Comparison between methods ipsogen JAK2 MutaScreen Kit and ARMS Results of ARMS testing method JAK2 V617F JAK2 V617F gt 2 lt 2 Total JAK2 V617F Mutation 91 91 detected Results of ipsogen JAK2 Inconclusive MutaScreen result testing JAK2 WT method No mutation detected Total 93 Table 20 Comparison between methods ipsogen JAK2 MutaScreen Kit and ARMS Agreement 95 CI 96 Positive data Agreement between ipsogen JAK2 98 9 94 1 99 8 MutaScreen Kit and ARMS Negative data Agreement between ipsogen JAK2 92 3 100 MutaScreen Kit and ARMS Total agreement 96 0 99 9 Confidence intervals were calculated according to CLSI EP12 A User Protocol for Evaluation of Qualitative Test Performance Approved Guideline Comparison between ipsogen JAK2 MutaScreen Kit and sequencing DNA samples from 51 patients with suspected MPN were tested in parallel with the ipsogen JAK2 MutaScreen Kit and the reference technique gold standard direct sequencing One sample could not be interpreted due to sequencing failure Comparisons of results obtained from the 50 interpretable samples are summarized in Table 21 2 x 3 contingency table and Table 2
28. any components not included within this Kit except as described in the ipsogen JAK2 MutaScreen RS Kit Handbook and additional protocols available at www qiagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated pe vue SED did The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com 2013 QIAGEN all rights reserved B Ft www giagen com Australia techservice au 2qiagen com Austria techservice at giagen com Belgium techservice bnl giagen com Brazil suportetecnico brasil giagen com Canada techservice ca giagen com China techservice cn Qqiagen com Denmark techservice nordic Qqiagen com Finland techservice nordic qiagen com France techservice fr Qqiag
29. arte aot MN 49 797 3 976 0 08 etecte MP 12 516 37 037 2 959 2 951 37 722 pond MP 12 958 38 121 2 942 efecte MI 54 394 6 39 0 117 0 119 1 516 Cut off sample MI 58 266 6973 0 12 M2 61 798 10 882 0 176 Ta DR Mutation M2 54 814 9 281 0 168 detected M3 57 364 16 604 0 289 Mutation M3 59 742 18 192 0 305 detected M4 56 965 28 99 0 509 rar ere Mutation M4 asy 2152 sop detected M5 54 251 37 221 0 686 sd a Mutation M5 54 979 36 125 0657 detected M6 46 185 44 498 0 963 NOR 125 Mutation M 45 077 42 598 0 945 detected S 13 47 37 409 2 777 2852 gedeg VU S 14 559 42 616 2 927 78 100 S2 50 432 24 958 0 495 Veen 0 505 6 46 2 53 797 27 746 0 516 12 5 31 S3 52 038 5 995 0 115 REOR 4 TN ER S3 54 0 6364 0 118 result S 4 QD 0 result GZ 1 425 1 607 44 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Troubleshooting guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see Contact Information page 54 Comments and suggestions Positive control signal negative a Pipetting error Check pipetting scheme and the setup of
30. ased erythropoiesis i e prefibrotic PMF 2 Not meeting WHO criteria for CML PV MDS or other myeloid neoplasm 3 Demonstration of JAK2V617F or other clonal marker or No evidence of reactive marrow fibrosis Minor 1 Leukoerythroblastosis 2 Increased serum lactate dehydrogenase LDH 3 Anemia 4 Palpable splenomegaly Recently international experts have proposed criteria for therapeutic trials in PV and ET Based on data on allograft alpha interferon or hydroxyurea JAK2V617F quantification has been incorporated as a potentially useful tool to monitor treatment response 9 A decrease in JAK2 V617F burden has been DE ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 5 observed in response to some of the new anti JAK2 targeted drugs in clinical development 10 Principle of the Procedure In an allelic discrimination assay two TaqMan probes are used in a multiplexed assay One is a perfect match to the allele 2 sequence e g the wild type allele the other one is a perfect match to the allele 1 sequence e g the allele with a mutation Each probe is labeled with a distinctive fluorescent dye at its 5 end the Reporter such as FAM or VIC and contains a non fluorescent Quencher at the 3 end The probes also contain a minor grove binder MGB permitting the use of shorter probes with greater stability and thereby a more accurate allelic discrimination During the extension phase of the PCR the perfectly matched
31. be 3 Prepare the following qPCR mix according to the number of samples being processed All concentrations are for the final volume of the reaction Table 12 describes the pipetting scheme for the preparation of one reagent mix calculated to achieve a final reaction volume of 20 ul A pre mix can be prepared according to the number of reactions using the same primer and probe mix Extra volumes are included to compensate for pipetting error On the LightCycler 2 0 instrument the ipsogen JAK2 MutaScreen RS Kit can be used for analysis of 7 samples in duplicate in one experiment Figure 27 Table 12 Preparation of qPCR mix for LightCycler 2 0 instrument Number of reactions pl Final Component 32 1 concentration LightCycler TaqMan Master Mix 5x oe I Primers and probes mix 10x Nuclease free PCR grade water 66 1x 297 Sample to be added at step 4 gt sach Total volume 20 each 14 samples 2 experiments kit ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 37 4 Vortex and briefly centrifuge the qPCR mix approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 5 Dispense 15 pl of the qPCR pre mix per capillary 6 Add 5 pl of the sample DNA material or controls in the corresponding capillary total volume 20 ul 7 Mix gently by pipetting up and down 8 Place the capillaries in the adapter provided with the instrument and briefly centrifuge 700 x g
32. ck Data then Convert and Next Select Comma and then click End The results will appear as shown in Figure 11 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 19 a e o o Ee Fr jseji jskitiMNiolPlalRis tlul viw t v ZI ANAE AC ADAE AF AGAH AL AJ AK AL AM AN AO AP AG ARASATIAUAVIAWAH ar A2 BA 1 Excel Raw Data Export 2 Copyright a QIAGEN Company All rights reserved 3 File Prog Allelic discrimination 2010 01 26 1 rex 4 Date 12612010 s Time inne 4 7 Channel Cycling A Green D MSPPOI 1 2 3 4I5EETEIHN 8 8 88H HH HH HR HH HH HH HH HH HH HH RR HEHEHE HH EE 49 50 s 1 pc 567432 581979 586158 5 91928 66 666666666666667777798 WW W AH amp W MH RN RN HH OR OH HHH HHH 63428 62 992195 2 pc 583912 59781 537958 6 02344 EEE Trike 547205 65 0160 o 3l nC 5 74617 5 79667 586391 581854 6 6 6 66 66 66 66 66 6666667777777778888888392392392394 999629 10 035624 12 4 nc 610742 6 40127 641794 641716 7777 77 7 2 2 72 2 2 2 2 7 7 78 8 8 8 88 8 9 9 9 4 d E 10 10 10 10 10 12 4178 12 607975 9 5 s 6 14824 6 24693 6 25727 6301066 66666 7 s 7888823239488 1 1l 1l s 8 4 amp 4 4 4 4 14 6672 14 944076 1 6 rs 5 869 5 93827 5 9459 5 98132 6 6 6 6 6 6 i 7788839392393999 9117 44944 13 0818 13 302488 O 5 7 h20 6 01612 6 13835 6 13298 6 16018 6 6 6 6 6 6 7777777777777777 7 77 7 740312 74200264 8 h20 5 77198 5 8514 5 90655 590239 6 6 6 6 6 6 77777777
33. crimination Figure 13 13 Accept the default settings for the Container and Template fields 96 Well Clear and Blank Document Figure 13 In the Plate ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 23 Name field type AD Post read Figure 13 and then click Next gt to access the Select Markers dialog box Hew Document Wizard Define Document Select the assay container and template for the document anu enter the operator name and comments 1 Assay Allelic Discrimination 00000 Container 3 ell Cear 2 Template li Blank Document Brosse Run Mode Standard 00 m F S00 Operator m aeee Comments 3 Plate Name aD Post read B 4 Figure 13 Pre settings for creating a new post read run New Document Wizard 14 If the Markers in Document panel in the Select Markers dialog box contains a suitable marker for your application proceed with step 18 If not then continue with step 15 15 Create detectors and markers as follows Click New Detector Figure 14 Hew Document Wizard Select Markers Select the markers you vill be using in the document Firal a Paserve Flefererce FO Marker Marne Detector 4 Markers in Document Add gt gt Hemove amp Finish Cancel Figure 14 The Markers in Document panel does not contain a suitable marker for your application 24 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 16
34. d ratio of the cut off sample M1 NRatio GZ NRatiom x 0 94 NRatioy x1 06 Compare the Normalized Ratio of each sample to the NRatio GZ Interpretation of results is outlined in Table 14 Table 14 Interpretation of genotyping results using normalized ratios Results Interpretation NRatiosgmpie gt NRoatioy x 1 06 JAK2 V617F is detected NRatiosgmpie lt NRatioy x 0 94 JAK2 V617F is not detected NRatiosgmple within NRatioy GZ Result inconclusive A semiquantitative result for the mutation burden can be obtained by comparing the value of each unknown sample ratio Ratiosgmpie with the mean ratio values of the reference scale Ratio 4 see Table 15 Table 15 Semiquantitative values for the JAK2 V617F mutation burden using the reference scale Results Mutation burden Ratio lt Ratiosgmple lt Ratioya 2 596 JAK2 V617F Ratioy lt Ratios lt Ratioys 5 12 5 JAK2 V617F Ratioy3 lt RatiOsgmple lt Ratio 12 5 31 JAK2 V617F Ratioya lt RatiOsample lt Rations 31 50 JAK2 V617F Ratioys lt RatiOsgmple lt Ration 50 7896 JAK2 V617F Ratioys lt RatiOsample 8 10096 JAK2 V617F An example of data calculation and interpretation is given in Table 16 Zo ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 43 Table 16 An example of fluorescence data calculation and interpretation using the reference scale Mean Sample VIC FAM Ratio ratio NRatio Interpretation MN 49 613 3 8 0 077 0 078 1 000 H
35. echnologies cat no 4304437 and LightCycler TaqMan Master Master Mix PCR 5x Roche cat no 04535286001 E Reagents for 0 8 196 agarose gel in 0 5x TBE electrophoresis buffer Consumables E Nuclease free aerosol resistant sterile PCR pipet tips with hydrophobic filters E 0 5 ml or 0 2 ml RNase and DNase free PCR tubes E Ice Equipment E Microliter pipets dedicated for PCR 1 10 ul 10 100 ul 100 1000 ul E Benchtop centrifuge with rotor for 0 2 ml 0 5 ml reaction tubes capable of attaining 10 000 rpm E Spectrophotometer for DNA quantitation E Real time PCR instrument Rotor Gene Q MDx 5plex HRM or other Rotor Gene instrument LightCycler 2 0 or 480 Applied Biosystems 7300 Real Time PCR System Applied Biosystems 7500 Real Time PCR System ABI PRISM 7000 SDS ABI PRISM 7700 SDS or ABI PRISM 7900HT SDS and associated specitic material E Equipment for pulsed field gel electrophoresis Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 9 Warnings and Precautions For in vitro diagnostic use When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com satety where you can find view and print the SDS for
36. ed data for all instruments Rotor Gene Q MDx 5plex HRM or other Rotor Gene instrument LightCycler 2 0 or 480 Applied Biosystems 7300 or 7500 Real Time PCR System ABI PRISM 7000 SDS 7700 SDS or 7900HT SDS and check the fluorescence levels these must be consistent between duplicates Prepare a graphical representation scatter plot of fluorescence data The x axis is VIC fluorescence the y axis is FAM fluorescence Graphical representation and quality control criteria An example of a scatter plot is shown in Figure 32 10 A MP i o H20 A MI 2 x M2 5 i x M3 12 m MA 31 M5 5076 B M6 78 e Positive samples Rn fluorescence FAM 530 nm O Negative samples A MN RN fluorescence VIC 560 nm Figure 32 Scatter plot of a representative allelic discrimination experiment Instruments Rotor Gene Q Applied Biosystems ABI PRISM and LightCycler 480 samples should be located on the arc connecting the negative controls MN to the positive controls MP Improper positioning of any control may indicate an experimental error E Positive controls should be located in the upper left E Negative controls should be located in the bottom right E Poor positioning of a negative control may indicate contamination S The cut off sample M1 of the reference scale should appear above the negative controls E Water controls should be located at the bottom left ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 41
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38. ep 1 50 0 c amp 0 30 Figure 18 Post read run Bo e ro o ees r oo lt rc m eoo 3 v e zz op r rr h _ UmDmu rvr1Pvzrir m h OBLAZU ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 27 27 Select File Export and then click Results to export the results to an Excel file The results will appear as shown in Figure 19 Comments VIC Sample 1 FAM Sample 1 SDS v1 2 sample Name Marker Task Passive Ref Allele X Allele Y Allele X Rn Allele Y R Quality Value Method sample 1 VIC Unknown 247 897 JAK2 VIC JAK2 FAM 2 184 5 221 Undetermined 100 00 Manual Call sample 1 VIC Unknown 295 555 JAK2 VIC JAK2 FAM 2 451 5 805 Undetermined 100 00 Manual Call sample 2 VIC Unknown 351 338 JAK2 VIC JAK2 FAM 2 585 6 2 Undetermined 100 00 Manual Call sample 2 VIC Unknown 379 909 JAK2 VIC JAK2 FAM 2 553 5 01 Undetermined 100 00 Manual Call sample 3 VIC Unknown 372 895 JAK2 VIC JAK2 FAM 2 913 5 329 Undetermined 100 00 Manual Call sample 3 VIC Unknown 359 717 JAK2 VIC JAK2 FAM 2 606 5 278 Undetermined 100 00 Manual Call sample wt VIC Unknown 343 535 JAK2 VIC JAK2 FAM 2 569 1 946 Undetermined 100 00 Manual Call sample wt VIC Unknown 277 577 JAK2 VIC JAK2 FAM 2 584 2 015 Undetermined 100 00 Manual Call C VIC Unknown 330 943 JAK2 VIC JAK2 FAM 2 523 1 967 Undetermined 100 00 Manual Call C VIC Unknown 314 523 JAK2 VIC JAK2 FAM 2 672 2 013 Undetermined 100 00
39. for the preparation of one reagent mix calculated to achieve a final reaction volume of 25 ul A pre mix can be prepared according to the number of reactions using the same primer and probe mix Extra volumes are included to compensate for pipetting error On Rotor Gene instruments the ipsogen JAK2 MutaScreen RS Kit can be used for analysis of 19 samples in duplicate in one experiment Figure 2 Table 3 Preparation of qPCR mix Number of reactions pl Final Component 56 1 concentration TaqMan Universal PCR Master Mix 2x A ds Primers and probes mix 10x Nuclease free PCR grade water 142 5 Ix 285 Sample to be added at step 4 gt sad Total volume 25 each 19 samples 1 experiment kit 4 Vortex and briefly centrifuge the qPCR mix approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 5 Dispense 20 pl of the qPCR pre mix per tube 6 Add 5 pl of the sample DNA material or controls in the corresponding tube total volume 25 pl 7 Mix gently by pipetting up and down 14 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 8 Close the PCR tubes Place the tubes in the 72 tube rotor according to the manufacturer s recommendations Fill all other positions with empty tubes 9 Make sure that the locking ring accessory of the Rotor Gene Instrument is placed on top of the rotor to prevent accidental opening of the tubes during the run Place the rotor in
40. icated material pipets tips etc in a dedicated area where no DNA matrixes DNA plasmid are introduced Add template in a separate zone preferably in a separate room with specitic material pipets tips etc 10 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Reagent Storage and Handling The kits are shipped on dry ice and must be stored at 30 C to 15 C upon receipt E Minimize exposure to light of the primers and probe mixes PPM VF tube E Gently mix and centrifuge the tubes before opening Store all kit components in original containers These storage conditions apply to both opened and unopened components Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results Expiration dates for each reagent are indicated on the individual component labels Under correct storage conditions the product will maintain performance until the expiration date printed on the label There are no obvious signs to indicate instability of this product However positive and negative controls should be run simultaneously with unknown specimens EZ 1L110 1 LUGLU4UUULLLUILUOULUULU L AAILLLULdXLI 1 LULULhI iLDA LX LUL EUNAALHAiL LELOLLLNLALALNNLLAAAALNLL LLLULLLAAAZZAZZAAO OUElM amp F K amp ZCLLLLLELLOOLIOLAUUZU ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 11 Procedure Sample DNA preparation Genomic DNA should be
41. isation Setup E x Auto Gain Optimisation Channel Settings Optimisation Ch IS Auto G ain Optimisation will read the fluoresence on the inserted sample at annel settings N different gain levels until it finds one at which the fluorescence levels are X e um acceptable The range of fluorescence you are looking for depends on the Channel Green Tube Position 1 ist Ss chemistry you are performing m m Target Sample Range Ib Flupb 10 Fl Set temperature to 4 degrees P 9 aid E u Optimise Acquiring w Perform Optimicatior Before 1st Acquisition Perform Optimisatior At 60 Degrees At Beginning Of Run Optimise All Acceptable Gain Range 10 to 10 4 Channel Settings Cancel Help TI a i Max Gain Edt Auto Gain Optimisation Channel Settings Green 1 5FI 10 Fl 10 10 m Yelow 1 5FI 10FI 40 10 Remove Channel Settings Remove All Channel Yellow Tube Position f Target Sample Range 5 Flup to 10 Fl Acceptable Gain Range 10 lo fo 4 Cancel Help Figure 6 Adjusting the fluorescence channel sensitivity 15 The gain values determined by the channel calibration are saved automatically and are listed in the last menu window of the programming procedure Click Start Run to run the program 16 Enter the rotor setup in the Rotor Gene software Figure 7 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 17 adit samples E 1 Eg File Edit Formak Security Setti
42. le Water control 2 reactions Sample processing on the LightCycler 480 instrument Figure 20 Suggested plate setup for an experiment with the ipsogen JAK2 MutaScreen RS Kit MP positive control MN negative control M1 to M6 reference scale S DNA sample H2O water control gt gt gt gt gt gt gt gt u lt vp n ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 29 qPCR on the LightCycler 480 instrument Note Perform all steps on ice Procedure 1 Thaw all necessary components and place them on ice Components should be taken out of the freezer approximately 10 minutes before starting the procedure 2 Vortex and briefly centrifuge all the tubes approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 3 Prepare the following qPCR mix according to the number of samples being processed All concentrations are for the final volume of the reaction Table 9 describes the pipetting scheme for the preparation of one reagent mix calculated to achieve a final reaction volume of 25 ul A pre mix can be prepared according to the number of reactions using the same primer and probe mix Extra volumes are included to compensate for pipetting error On the Applied Biosystems 7300 and 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments the ipsogen JAK2 MutaScreen RS Kit can be used for analysis of 19 samples in duplicate in one experiment Figu
43. left and click gt To stop acquiring from a Click on one of the steps below to modify it or press or to add and remove steps for this cycle T Dap aang channel select it in the right hand list and click lt To remove all acquisitions click lt lt 92 deg for 15 secs NN EL oc NIE Not Acquaring Dye Chart gt gt Don t Acquire Help 6 Dye Channel Selection Chart Cc II Sour Detector D 60 deg for 60 secs Green 470nm 510nm Fam SYBR Green 1 Fluorescein EvaGreen Alexa Fluor 488 ee cree Long Range Touchdown Yellow 530m 555nm JDE 4 VIC HEX TET CAL Fluor Gold 540 Yakima Yellow ROX CAL Fluor Red 610 Cy35 Texas Ried Alexa Fluor 568 4 Cy5 Quasar 670 Alexa Fluor 633 Crimson 710hp Quasar705 Alexa Fluor 680 Figure 5 Amplification of the DNA 14 The detection range of the fluorescence channels has to be determined according to the fluorescence intensities in the PCR tubes Click Gain Optimisation in the New Run Wizard dialog box to open the Auto Gain Optimisation Setup dialog box Click Optimise Acquiring Figure 6 and then click OK in the Auto Gain Optimisation Channel Settings dialog boxes for each channel Green and Yellow Figure 6 Make sure that the Perform Optimisation Before 1st Acquisition box is checked for each channel Figure 6 Auto Gain Optim
44. lele X and FAM fluorescence for Allele Y Figure 24 Create new analysis Allele Y Hex 523 568 Figure 24 Selecting fluorescence for Allele X and Allele Y 16 The next window Figure 25 shows plate setup 1 upper left fluorescence results for each sample 2 bottom left and the scatter plot with allelic discrimination 3 right FAM and VIC fluorescence measured at the 50th PCR cycle a 2 123 533 Samples Endpoint Fluorsecence Pes Mame 523568 3 533 1004 2 10 97 46 354 amp 1004 2 0 30 0 38 1004 2 0 37 0 43 EZO 0 21 0 29 0 23 0 32 u zu FEI 5 15 40 27 13 12 2 26 80 17 38 32 03 12 50 33 28 14 09 33 26 14 76 34 58 38 54 32 55 JS rY 33 26 du 5000 1000 1500 20000 25000 30000 35000 40 00 32 79 25 03 Flunreseenee 523 558 27 78 Hew Call Apply Color Comp Filter Comb Analysis v on LY nm sron iv asse raso Maier IV j I E NE EM fF jf ESI BH NE EE EH Figure 25 Data summary 17 To export data right click on the sample results template and then select Export Table The file will be saved in a text txt file format 34 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 18 To view and analyze results open the file using Excel The results will appear as shown in Figure 26 Microsoft Excel test 3 Fichier Edition Affichage Insertion Format utils Donn es Fen tre D c E ah MOBO o u fret MM Ba
45. ler 2 0 instrument Note Because of particular technological requirements LightCycler 2 0 experiments must be performed using specific reagents We recommend the use of the LightCycler TaqMan Master Follow the manufacturer s instructions to prepare the Master Mix 5x Using a 32 capillary rotor we recommend performing all measurements in duplicate as indicated in Table 11 Table 11 Number of reactions for LightCycler 2 0 instrument Samples Reactions JAK2 V617F primers and probes mix PPM VF 32 reactions 7 DNA samples 7 x 2 reactions 2 x 2 reactions MP VF MN VF each one tested in duplicate 6 x 2 reactions M1 to M6 each one tested in duplicate 2 DNA controls Reference scale Water control 2 reactions Sample processing on LightCycler 2 0 instrument H O M M H O M2 MN ie 06 M2 Figure 27 Suggested rotor setup for an experiment with the ipsogen JAK2 MutaScreen RS Kit MP positive control MN negative control M1 to M6 reference scale S DNA sample H2O water control 36 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 qPCR on LightCycler 2 0 instrument Note Perform all steps on ice Procedure 1 Thaw all necessary components and place them on ice Components should be taken out of the freezer approximately 10 minutes before starting the procedure 2 Vortex and briefly centrifuge all the tubes approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tu
46. ngs Given Conc Format 123456 7831 23467 c Limit copies reactio More Options Samples H ID Mame Type Groups Given Cone Ah 1 PC Positive Control Im 2 PC Positive Control BM H20 NTC 4 Mone E 5 NC Negative Control a B NC Negative Control EN H20 NTC a Mone 3 3 AS Unknown BU ions Unknown 11 Mone 12 Mone 13 57 LInknown 14 51 Unknown 15 52 Unknowr 16 52 LInknown 17 53 Unknown 1853 Unknowr 19 54 Unknowr Unknown RE k VN AKZ Ipsogen Ep Eu Heu Synchronize pages BEER na e un n T n rm m Undo OF Cancel Help Figure 7 Rotor Gene setup Edit Samples End point analysis procedure for Rotor Gene Q 5plex HRM instrument setting 17 After the PCR program has ended click Analysis in the toolbar Figure 8 B Analysis Only Rotor Gene Q Series Software VIRTUAL MODE Prog Allelic discrimination 2010 01 26 1 File Analysis Gain View Window Help m ir r i t l l x View GP Fig Fi Wig ex zum f In Open Save Help Settings Profile Temp Samples Analysis Reports Arrange Channels Cycling 4 Green Cycling A Yelow Cycling B Green 2 Cycling B Yellow VS Figure 8 Analysis 18 In the Analysis dialog box Figure 9 double click Cycling A Green and then OK Repeat for Cycling A yellow 18 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Analysis x 2 Std Cu
47. obtained either from whole blood purified peripheral blood lymphocytes polynuclear cells or granulocytes To be able to compare results we recommend adopting the same cellular fraction and DNA extraction method DNA extraction should be performed by any home brew or commercial method DNA quantity is determined by measuring optical density at 260 nm DNA quality should be assessed by spectrophotometry or gel electrophoresis The Asso Asgg ratio should be 1 7 1 9 Smaller ratios usually indicate contamination by protein or organic chemicals Electrophoretic analysis on a 0 8 196 agarose gel should allow visualization of the isolated DNA as a distinct band of about 20 kb A slight smear is acceptable The resultant DNA is diluted to 5 ng ul in TE buffer The qPCR reaction is optimized for 25 ng of purified genomic DNA Storing nucleic acids For short term storage of up to 24 hours we recommend storing purified nucleic acids at 2 8 C For long term storage of over 24 hours we recommend storage at 20 C Protocol qPCR on Rotor Gene Q instruments with 72 tube rotor Using this instrument we recommend performing all measurements in duplicate as indicated in Table 2 Table 2 Number of reactions for Rotor Gene Q MDx 5plex HRM or Rotor Gene Q 5plex HRM instruments with 72 tube rotor Samples Reactions JAK2 V617F primers and probes mix PPM VF 56 reactions 19 DNA samples 19 x 2 reactions 2 x 2 reactions MP VF MN VF each
48. olume 25 each 19 samples 1 experiment kit 22 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 4 Vortex and briefly centrifuge the qPCR mix approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 5 Dispense 20 pl of the qPCR pre mix per well 6 Add 5 pl of the sample DNA material or controls in the corresponding well total volume 25 ul 7 Mix gently by pipetting up and down 8 Close the plate and briefly centrifuge 300 x g approximately 10 seconds 9 Place the plate in the thermal cycler according to the manufacturer s recommendations 10 Program the thermal cycler with the thermal cycling program as indicated in Table 7 and start the run Table 7 Temperature profile for Applied Biosystems and ABI PRISM instruments Hold Temperature 50 C Time 2 minutes Hold 2 Temperature 95 C Time 10 minutes Cycling 50 times 92 C tor 15 seconds 60 C for 1 minute Post read run analysis procedure for Applied Biosystems and ABI PRISM instruments For programming details of the Applied Biosystems 7300 and 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments refer to the instrument user guide For a better overview the software settings are framed in bold black 11 After the run is finished select Start Program and then select File New 12 In the New Document Wizard dialog box click the Assay drop down list and select Allelic Dis
49. ory analytical data A multi center study was performed involving 13 laboratories Analytical data were collected on dilutions of genomic DNA harboring JAK2 V617F mutation in wild type DNA Three experiments were performed in each laboratory For each experiment the following DNA samples were tested from cell lines E 1 negative control NC 096 V617F E 1 positive control PC 10096 V617F E 1 cut off sample COS 2 V617F E 3 samples harboring intermediate mutation loads 20 5096 and 80 The experiments were performed on 7 different instrument models ABI PRISM 7000 SDS 11 Applied Biosystems 7300 Real Time PCR System 1 Applied Biosystems 7500 Real Time PCR System 3 ABI PRISM 7700 SDS 11 ABI PRISM 7900 SDS 1 LightCycler 2 0 1 and iCycler 1 Results are summarized in Table 18 Table 18 Interlaboratory analytical data obtained from dilutions of genomic DNA from cell lines harboring the JAK2 V617F mutation in wild type DNA Sample detection Positive samples Negative samples JAK2 V617F 177 0 JAK2 wild type O 36 Positive samples included 36 positive controls PC 36 cut off samples COS 2 V617F 34 samples harboring 20 JAK2 V617F 35 samples harboring 50 JAK2 V617F and 36 samples harboring 80 JAK2 V617F Clinical studies Comparison between ipsogen JAK2 MutaScreen Kit and ARMS method DNA samples from 141 patients with suspected MPN were tested in parallel with the ipsogen JAK2 MutaScreen Kit
50. p E Am eosmseme 14 24 25 Estimated Tame him ss Figure 22 LightCycler 480 II Setting the detection format 32 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 12 Program the thermal cycler with the thermal cycling program as indicated in Table 10 and start the run Note When describing the plate setup on the instrument select Endpt Geno in the Step 1 select workflow section Table 10 Temperature profile for the LightCycler 480 instrument Hold Temperature 50 C Time 2 minutes Hold 2 Temperature 95 C Time 1O minutes Cycling 50 times 92 C tor 15 seconds single 60 C for 1 minute single 60 C for 1 minute single End point analysis procedure for the LightCycler 480 instrument 13 After the run is finished click Analysis 14 In the Create New Analysis dialog box select Endpoint Genotyping and then select the subset to analyze in the Subset menu Figure 23 Create New Analysis Create new analysis Subset 2 Program Abs Quant 2nd Derivative Max Abs Quant Fit Points Advanced Relative Quantification Basic Relative Quantification Color Compensation MEndpoint Genotyping Melt Curve Genotyping Tm Calling Name 2 B a oo OD A n o T IF ol W Figure 23 Selecting analysis type and subset to analyze ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 33 15 In the next window select Hex i e VIC fluorescence for Al
51. plete the fields clicking Next when you are ready to move to the next page Operator be Notes Programme PCR allelic discrimination J Reaction 25 E Volume pL Ex Sample Layout el C J oo LET Figure 4 Setting the general assay parameters 13 Click the Edit Profile button in the next New Run Wizard dialog box and program the temperature profile as shown in Table 4 and Figure 5 Be sure to add the last acquiring step at 60 C at each cycle for both channels Green FAM and Yellow VIC Table 4 Temperature profile Temperature 50 deg Time 2 mins Temperature 95 deg Time 10 mins 50 times 92 deg for 15 secs 60 deg for 1 min single Acquisition of FAM fluorescence in channel Cycling A Green Acquisition of VIC fluorescence in channel Cycling A Yellow 16 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 i Edit Profile oe T H 7 New Open SaveAs Help The run will take approximately 110 minute s to complete T he graph below represents the run to be performed y Acquisition Same as Frevious New Acquisition Acquistion Configuration Available Channels Acquiring Channels Click on a cycle below to modify it Name gt Hold Insert after Crimsora Orange lt g Yelow Cycling Insert before Red 7 5 Remove This cycle repeats N 2A 50 J time s To acquire from a channel select it from the Est in the
52. r on the packaging and labeling Ny EN Contains reagents sufficient for lt N gt reactions gt lt Use by IVD In vitro diagnostic medical device Catalog number Lot number MA Material number Temperature limitation Manufacturer Consult instructions for use Ea es NN Contact Information For technical assistance and more information please see our Technical Support Center at www qiagen com Support call 00800 22 44 6000 or contact one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com 54 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 Ordering Information Product Contents Cat no ipsogen JAK2 For 19 reactions V617F Positive 673123 MutaScreen RS Kit 19 Control V617F Negative Control V617F Reference Scale Primers and Probes Mix JAK2 wild type and JAK2 V617F Rotor Gene Q MDx for IVD validated real time PCR analysis in clinical applications Rotor Gene Q MDx Real time PCR cycler and High 9002032 5plex HRM Plattorm Resolution Melt analyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories 1 year warranty on parts and labor installation and training not included Rotor Gene Q MDx Real time PCR cycler and High 9002033 5plex HRM System Resolution Melt analyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories 1
53. re 20 Table 9 Preparation of qPCR mix Number of reactions pl Final Component 56 1 concentration TaqMan Universal PCR Master Mix 2x War x Primers and probes mix 10x Nuclease free PCR grade water 142 5 Ix 285 Sample to be added at step 4 gt Gen Total volume 25 each 19 samples 1 experiment kit 30 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 4 Vortex and briefly centrifuge the qPCR mix approximately 10 seconds 10 000 rpm to collect the liquid in the bottom of the tube 5 Dispense 20 pl of the qPCR pre mix per well 6 Add 5 pl of the sample DNA material or controls in the corresponding well total volume 25 ul 7 Mix gently by pipetting up and down 8 Close the plate and briefly centrifuge 300 x g approximately 10 seconds 9 Place the plate in the thermal cycler according to the manufacturer s recommendations 10 On the home page select New Experiment 11 For the LightCycler 480 I follow step 11a For the LightCycler 480 II follow step 11b For programming details of the LightCycler 480 instrument refer to the instrument user guide For a better overview the software settings are framed in bold black 11a LightCycler 480 I Select Multi Color Hydrolysis Probe click Customize and then check that the channels FAM 483 533 and Hex 533 568 i e VIC are selected Figure 21 Set the reaction volume to 25 ul Figure 21 and proceed with s
54. retation of Results 41 Graphical representation and quality control criteria 4 Calculation of normalized FAM VIC ratio and genotyping 42 Troubleshooting guide A5 Quality Control 47 Limitations 47 Performance Characteristics 47 Nonclinical studies 47 Clinical studies 48 References 53 Symbols 54 Contact Information 54 Ordering Information 55 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 3 Intended Use The ipsogen JAK2 MutaScreen RS Kit is intended for the detection of JAK2 V617F G1849T mutation in genomic DNA from subjects with suspected myeloproliferative neoplasm The absence of JAK2 V617F G1849T does not exclude the presence of other JAK2 mutations The test can report false negative results in case of additional mutations located in codons 615 to 619 1 Note The kit should be used following the instructions given in this manual in combination with validated reagents and instruments Any off label use of this product and or modification of the components will void QIAGEN s liability Summary and Explanation A recurrent somatic mutation V617F affecting the Janus tyrosine kinase 2 JAK2 gene has been identified in 2005 2 5 leading to a major breakthrough in the understanding classification and diagnosis of myeloproliferative neoplasms MPN JAK2 is a critical intracellular signaling molecule for a number of cytokines including erythropoietin The JAK2 V617F mutation is detected in gt 95 of patients with polycythemi
55. rves Rel Other Cycling A Yellow At Least Two Standards Required d q The auto find threshold feature requires that vou have defined at least 2 selected standards To set this up right click on the sample list and select E dit Samples Don t show this message in future Show Hide Auto shrink window Figure 9 Quantitation Cycling A Green 19 A new window appears Figure 10 Click Slope Correct in both panels as shown in Figure 10 oH m Q jw d Sf BN Ww Ww Oc hated Open ET Setting Progress Profile Temp Samples nae Repris Annan Chanmets pirg A Gneen j Cycding A Yellow 5 Quantitation Analysis Cycling A Yellow imspp ez 7 A del SEE 2 il Adjust Scale Atoka Defeut Scale linear Scale i Ho Name Toe m Given Conc cop Cso Conc copie X Wer Rep Ct No Mame a pet Panes C l 1 po wt F fe wt Peut C E Bi 3 mew Negatnes 3 ne ud 4 ric wt Hegatree 3 m wt 5 Unkresen 5 eos amp Urrira 15 eni h HTC hin B ke HTC j ken 3 Linkr Em I gl Li rire 0 exe 1 0 Unies jn 15 OB Uniram 12 5x 7 113 1 Unkresen 113 1 Undunosen I Insert Rew Date EI of lei gt Ihoesheid xj Figure 10 Setting Slope Correct 20 To export data save as an Excel data sheet Click OK give a name to the export file and save the text file txt 21 Open the text file in Excel and select column A Cli
56. ry i E I L ni Lh D PILLI TIPINI TT TFETIWITTTYTETIMITE TTITITTETT H bitii asa p44 ian IHH g md amp xp m RESET ETT faves ere ume fare cee d EDT SHEERS SERE SRZRZER ERE n a Damgan abo j T 5 bi SHEE S Figure 29 Results and history in Online Data Display 12 Right click near the Current Fluorescence graph and select Export ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 39 13 Click the Excel box on the Export chart dialog box Figure 30 Enter a name in the Filename dialog field Select an export destination for the result file with the button Click Export End Program 10 Cycles Color Comp Off Instrument Name LC 5312 ID LC 6812 Online Data Display Run Notes Jlete Samples Bb Export chart os Name 1 Sample Picture Data Jal x 2 Sample Series all Include 3 Empty P IV Point Index 4 Sample Format 7 Point Labels 5 Sample Text V Header A Fa C XML H ample 1 8 Sample s HTM Table Delimiter nplc a Tab Y Figure 30 Selecting the export format and data file destination 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18 Sample 23 Sample 24 Sample ar 14 To view and analyze results open the file in Excel The results for LightCycler 2 0 will appear as shown Position X Bar Text X Bar Text X Text 123
57. tep 12 emper ature Targets E Target C Acquisition Mode Hold ihhzmancss Ramp Rate fe Acquisition per C Sec Tanger C Step Size C Step Delay cyclen Reno kien Lene 00 04 00 4 4 jo 10 Jo e e gt ut Detection Formats E E ei Acti Filter Combinati S Bo vir Zn oy pos FAR 4653 534 3 f m Hex 523 860 z EB 610 4 35 oy 9 615 670 oo e OE QC ee bar 106 Estimated Tiet limes Apply v Y Figure 21 LightCycler 480 I Setting the detection format ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 31 11b LightCycler 480 Il Select Dual Color Hydrolysis Probe click Customize and then check that the channels FAM 465 510 and VIC HEX 533 580 are selected Figure 22 Set the reaction volume to 25 ul Figure 22 and proceed with step 12 5 v Instrument Virtual LightCycler 480 96 System Het Connected Datal My Computer Research Window u System Ad I 2 1 E Customize 4 Sulizet z Color Comp ID Editor me Programs Sample Program Hame Cycles Analysis Mose Editar e k Program 1 Hone Detection Formats Detection Format Dual Color Hydrolysis Probe UPL Probe Target C E lt O gt KO gt Step Size lC Step Delay cycles i in a LI ALLL 1 n b 10 FAH 4655 510 VIC HEX Yellow555 533 580 QJ Temperature C E Em 8 2 E am
58. the Rotor Gene Q instrument according to the manufacturer s recommendations 10 For the detection of JAK2 DNA create a temperature profile according to the following steps Setting the general assay parameters Figures 3 4 Amplification of the DNA Figure 5 Adjusting the fluorescence channel sensitivity Figure 6 Please find further information on programming Rotor Gene Instruments in the instrument user manual In the illustrations the software settings are framed in bold black Illustrations are included for Rotor Gene Q Instruments 11 Start the Rotor Gene Software In the New Run dialog box click New Hew Run ES Quick Statt Advanced d j Imports khe cvcling Perform Last Run j and acquisition and sample definitions Empty Run From the last run open in the software Three Step with Melt Two Step V M HRM V Other Runs Instrument Maintenance j Open 4 Template In Another Folder I Mew ancel Help W Show This Screen when Software Opens Figure 3 The New Run dialog box ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 15 12 In the New Run Wizard set the volume to 25 ul and click Next Mew Run Wizard This bos displays help on elements in the wizard For help on an item haver your mouse over the item for help rau can alzo click on a combo box to display help about its available settings This screen displays miscellaneous options for the run Com
59. w ae a Al Experiment OB 08 12 16 Active filters FAM 433 533 Hex 523 568 A B C D E F G VIC 1 Esperiment ob 08 12 16 Active filters FAM 483 533 Hex 523 568 FAM 2 include Color Pos Name 523 568 Score 3 True 10789024 AS 100 20 10 971 0 00 4 True 107390243 A6 l0096 420 0 302 0 00 5 True 10739024 A7 100 20 0 369 0 00 6 True 10789024 ALO H20 0 207 0 00 J True 10 29024 ALI H20 0 222 0 00 8 True 10789024 AL2 Hz2O0 0 203 0 00 9 True 10789024 B5 7896 20 25 731 0 00 10 True 107389024 B6 7898 20 27 125 0 00 1l True 10789024 B7 7896 20 26 803 0 00 12 True 10789024 C5 50 20 32 035 0 00 13 True 10785024 C 5096 20 33 278 0 00 14 True l10 39024 Ce 5096 20 23 2601 0 00 15 True 107389024 DS 3196 20 34 534 0 00 16 True 10785024 D6 31 20 32 549 0 00 17 True 10789024 D7 31 20 33 262 0 00 18 True 10789024 E5 12 575 20 32 794 0 00 19 True 10785024 E6 12 596 20 34 932 0 00 20 True 10785024 E7 12 596 20 35 089 0 00 21 True 10739024 FS 576 20 35 838 0 00 22 True 10789024 F6 576 20 36 756 0 00 23 True 10785024 F7 556 20 36 546 0 00 24 True 10789024 G5 296 20 35 082 0 00 25 Irue 10739024 G6 fre 20 25 834 0 00 26 True 10789024 G7 2 70 34 299 0 00 27 True 10759024 HS 0 e 20 34 449 0 00 28 True 10 29024 H6 gt 20 33 520 0 00 29 True 10789024 H7 5 20 34 125 0 00 Figure 26 Example of results shown in an Excel file ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 35 Protocol qPCR on LightCyc
60. year warranty on parts and labor installation and training For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 55 This page intentionally left blank 56 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 This page intentionally left blank ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 57 This page intentionally left blank 58 ipsogen JAK2 MutaScreen RS Kit Handbook 01 2013 This product is intended for in vitro diagnostic use ipsogen products may not be resold modified for resale or used to manufacture commercial products without written approval of QIAGEN Information in this document is subject to change without notice QIAGEN assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall QIAGEN be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document ipsogen products are warranted to meet their stated specifications QIAGEN s sole obligation and the customer s sole remedy are limited to replacement of products free of charge in the event products fail to perform as warranted

Sample & Assay Technologies ipsogen® JAK2

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Sample &amp; Assay Technologies ipsogen&reg; JAK2

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Sample & Assay Technologies ipsogen® JAK2