ipsogen® JAK2 MutaQuant® Kit Handbook
Contents
1. reagents of the reaction Repeat the PCR run ipsogen NPM1 MutaScreen Handbook 01 2013 25 Comments and suggestions c Inappropriate storage of kit Aliquot reagents for storage components Store the ipsogen NPM1 MutaScreen Kit at 15 to 30 C and keep primers and probe mixes protected from light See Reagent Storage and Handling page 8 Avoid repeated freezing and thawing d Incorrect detection channel has Set channel to Green channel or been chosen 530 nm 640 nm e No data acquisition program Check the cycle program Select acquisition mode Single at the end of each annealing segment of the PCR program Fluorescence intensity varies a Pipetting errors Check pipetting scheme and the setup of the reaction Vortex and spin all reagents after thawing Repeat the PCR run b Insufficient centrifugation of the Always centrifuge tubes or plates tubes or plate loaded with the reaction mix as described in the specific operating manual of the apparatus Inconsistent C values between duplicates a Pipetting errors or cross Check pipetting scheme and the setup contamination of the reaction Repeat the PCR run b Low mutation load Always check the DNA quality OD 69 OD229 and concentration before starting 26 ipsogen NPM1 MutaScreen Handbook 01 2013 Comments and suggestions Fluorescence intensity is too low a Inappropriate storage of kit Aliquot reagents for storage components Store the2. 2 32 73 32 78 Undetected Undetected MutA 3 29 12 Undetected 29 79 Undetected MutB 4 34 92 Undetected 33 57 Undetected MutB 27 49 36 28 Undetected 27 96 MutD 6 33 73 Undetected Undetected 33 54 MutD 7 27 06 37 23 38 7 Undetected Not typed 8 27 87 38 91 Undetected Undetected Not typed 9 Undetected Undetected Undetected Undetected NA If a sample has C values for more than one of the primers and probe mixes for detecting MutA MutB or MutD mutations the lowest of these C values for the sample is used to assign the mutation type See sample 1 and sample 5 in Table 12 Samples may be genotyped providing the conditions of Rule 7 are met Conditions of Rule 7 have been met in 6 of 8 samples Rule 7 The NPM1 mutation type of a sample is determined by the lowest C value obtained with PPM NPM1 MutA PPM NPM1 MutB and PPM NPM1 MutD providing Cy NPM1 MutA MutB or MutD Sample lt Cy mu nemi Sample 4 4 For sample 7 and sample 8 the Cy Nemi muta Sample values 37 23 and 38 91 are greater than the Cy mutNem1 Sample values 4 4 These values are 31 46 and 32 27 respectively 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 i
3. MutB and PPM NPM1 MutD The mutation type is indicated by the result with the lowest C value providing that C value respects the following rule Cy NPM1 MutA MutB or MutD Sample lt Cr mur nemi Sample 4 4 A summary of the rules of sample validation and analysis is given in Table 8 Table 8 Successive rules applied for run validation and results analysis No Stage Rule wales Must give zero C values controls 2 NPMI wild Must be detected with only Quality type control PPM total NPM1 trol Zu Mutated 3 NPMI Must be detected with all primer and positive probe mixes control 4 Input C Total npm WTC C Total NPM1 Mut PC 2 3 Sample interval 5 input Sample The value of C Total nem Sample must be validation within the input interval En A sample is positive for an NPMI l 6 en mutation if a Cy value is obtained with PPM Mut NPM1 Cr munem Sample The NPM1 mutation type of a sample is Analysis determined by the lowest C value Mutation obtained with PPM NPM1 MutA PPM 7 type NPM1 MutB and PPM NPM1 MutD detection providing Cy NPM1 MutA MutB or MutD Sample lt C4 mutNpm1 Sample 4 4 20 ipsogen NPM1 MutaScreen Handbook 01 2013 Step by step interpretation of results Interpretation is shown of results from 9 samples plus controls analyzed with the ipsogen NPM1 MutaScreen Kit Quality control Quality control using C values of controls is shown in Table 9 Table 9 Run validation o
4. With the NPM1 MutA primers and probe mix PPM NPM1 MutA With the NPM1 MutB primers and probe mix PPM NPM1 MutB With the NPM1 MutD primers and probe mix PPM NPM1 MutD n DNA samples n x 2 reactions 4 reactions wild type control WTC and mutated positive control Mut PC each one tested in duplicate 2 DNA controls Water control 2 reactions Sample processing on Rotor Gene Q instruments with 72 tube rotor We recommend testing 4 DNA samples in the same experiment 14 reactions with each primers and probe mix to optimize the use of controls and the primers and probe mixes a ee 10 ipsogen NPM1 MutaScreen Handbook 01 2013 Mut g cl 09 en Mut He sl 1 52 52 3 me ie WTC 2 ey RAG 54 H0 e oo e O OOo H20 or Se S O O WTC s4 O i O PPM Total NPM 1 oo 53 MutPC ve 52 O S1 S lt 52 1 PPM Mut NPM 1 O 2 s Foes A PPM NPM1 Mut D p MutPC 7 Ss WTC 2 wir O no H20 O Ho a PPM NPM 1 Mut A WIC WTC i O MutPC amp MutPC O 5 Oo 90 a l Mut OO O OO 53 ir Pe WIG H20 H20 54 a Figure 3 Suggested rotor setup for an experiment with the ipsogen NPM1 MutaScreen Kit WTC wild type NPM1positive control Mut PC mutated NPM1 positive control S DNA sample H20 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 i
5. free PCR grade water E Buffer and Tag DNA polymerase The recommended reagents are TaqMan Universal PCR Master Mix Master Mix PCR 2x Life Technologies cat no 4304437 Consumables M Nuclease free aerosol resistant sterile PCR pipet tips with hydrophobic filters BE 0 5 ml or 1 5 ml nuclease free PCR tubes E Ice Equipment E Microliter pipet dedicated for PCR 1 10 ul 10 100 ul 100 1000 ul E Benchtop centrifuge with rotor for 0 5 ml 1 5 ml reaction tubes and microplates capable of attaining 13 000 14 000 rpm E Real time PCR instrument Rotor Gene Q 5plex HRM or other Rotor Gene instrument LightCycler 480 Applied Biosystems 7500 Real Time PCR System ABI PRISM 7900HT SDS and associated specific material M Spectrophotometer Warnings and Precautions When working with chemicals always wear a suitable laboratory 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 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 Ensure that instruments have been che
6. sample Quality control using C values of controls The NPMI wild type control WTC and the mutated NPMI positive control Mut PC allow an experiment to be qualified EHE The NPMI wild type control must be detected with only PPM Total NPMI E The mutated NPMI positive control must be detected with all PPMs The entire experiment is rejected if both conditions are not met Water controls Water controls non template controls should give zero C values for all primers and probe mixes A positive water control results from a cross contamination See Troubleshooting guide below to find a solution Sample input validation A sample s input must be validated before interpretation The validation interval is determined with these calculations Cr Total npm WTC C value of the NPM1 wild type control with PPM Total NPM1 Cy Total npm Mut PC C value of the mutated NPMI positive control with PPM Total NPM1 C Total PMI Sample C value of a sample with PPM Total NPMI The value of C Total npm1 Sample must be within the following interval Cy Total npm WTC Cr Total nemi Mut PC 2 3 The sample is positive for an NPM1 mutation if a C value is obtained with the primers and probe mix PPM Mut NPM1 This is Cr mut nem Sample ipsogen NPM1 MutaScreen Handbook 01 2013 19 The NPM1 mutation type of a sample mutA MutB or MutD is determined by the C values obtained with PPM NPM1 MutA PPM NPM1
7. 60 C for 1 minute 30 seconds with acquisition of FAM fluorescence quencher TAMRA Table 7 Temperature profile for LightCycler 480 instrument Mode of analysis Absolute Quantification Abs Quant Detection formats Select Simple Probe in the Detection formats window Hold Temperature 50 C Time 2 minutes Hold 2 Temperature 95 C Time 10 minutes Cycling AO times 95 C for 15 seconds 60 C for 1 minute 30 seconds with acquisition of FAM fluorescence corresponding to 483 533 nm for LC version 01 and 465 510 nm for LC version 02 ipsogen NPM1 MutaScreen Handbook 01 2013 17 9 For the Applied Biosystems 7500 and ABI PRISM 7900HT instruments follow step 9a For the LightCycler 480 instrument follow step 9b 9a Applied Biosystems 7500 and ABI PRISM 7900HT instruments We recommend a threshold set at 0 1 and a baseline set between cycles 3 and 15 Start the cycling program as indicated in Table 6 9b LightCycler 480 We recommend a Fit point analysis mode with background at 2 0 and threshold at 2 0 Start the thermal cycling program as indicated in Table 7 18 ipsogen NPM1 MutaScreen Handbook 01 2013 Results Calculate the mean threshold cycle C value obtained with each primers and probe mix for the controls NPM1 wild type control and mutated NPM1 positive control and for each sample If one of the duplicates of a sample has an undetermined value we recommend retesting the
8. January 2013 ipsogen NPM1 MutaScreen Handbook R For detection of NPM1 mutations and identification of mutation types A B and D For research use only Not for use in diagnostic procedures For use with Rotor Gene Q Applied Biosystems 7500 Real Time PCR System ABI PRISM 7900HT SDS and LightCycler 480 instruments CREF 677013 eal QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden GERMANY 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 Principle of the Procedure 4 Materials Provided 6 Kit contents 6 Materials Required but Not Provided 6 Warnings and Precautions 7 General precautions 8 Reagent Storage and Handling 8 Procedure 9 Sample DNA preparation 9 Protocols E gPCR on Rotor Gene Q 5plex HRM instruments with 72 tube rotor 10 E gPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments and LightCycler 480 instrum
9. ated positive control Mut PC each one tested in duplicate 2 DNA controls Water control 2 reactions Sample processing on Applied Biosystems 7500 and ABI PRISM 7900HT instruments and LightCycler 480 instrument We recommend testing 4 DNA samples in the same experiment 14 reactions with each primers and probe mix to optimize the use of controls and the primers and probe mixes a 14 ipsogen NPM1 MutaScreen Handbook 01 2013 Figure 4 Suggested plate setup for an experiment with the ipsogen NPM1 MutaScreen Kit WTC wild type NPMI positive control Mut PC mutated NPM1 positive control S DNA sample H20 water control S DNA sample H O water control qPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments and LightCycler 480 instrument Note Perform all steps on ice Procedure 1 Thaw all necessary components and place them on ice 2 Prepare the following qPCR mix for each primers and probe mix according to the number of samples being processed All concentrations are for the final volume of the reaction Table 5 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 PPM Total NPM1 PPM Mut NPM1 PPM NPM1 MutA PPM NPMI MutB or PPM NPM1 MutD Extra volumes are included to compensate for pipetting error i
10. cked and calibrated according to the manufacturer s recommendations ipsogen NPM1 MutaScreen Handbook 01 2013 7 General precautions Use of qPCR tests require good laboratory practices including maintenance of equipment that are dedicated to molecular biology and is compliant with applicable regulations and relevant standards This kit is intended for research use Reagents and instructions supplied in this kit have been tested for optimal performance Further dilution of the reagents or alteration of incubation times and temperatures may result in erroneous or discordant data Primers and probe mix PPM reagents may be altered if exposed to light All reagents are formulated specifically for use with this kit For optimal performance of the kit no substitutions should be made Use extreme caution to prevent E DNase contamination that might cause degradation of the template DNA M 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 HE Use fresh aerosol resistant pipet tips for all pipetting steps to avoid cross contamination of the samples and reagents M Prepare pre PCR master mix with dedicated material pipets tips etc in a dedicated area where no DNA matrices DNA plasmid or PCR products are introduced Add template in a separate zone preferably in a
11. ent 14 Results 19 Step by step interpretation of results 21 Troubleshooting guide 24 Quality Control 28 References 28 Symbols 28 Contact Information 28 Ordering Information 29 ipsogen NPM1 MutaScreen Handbook 01 2013 3 Intended Use The ipsogen NPM1 MutaScreen Kit is intended for research use only Not for use in diagnostic procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Principle of the Procedure The ipsogen NPM1 MutaScreen Kit combines two techniques to screen for the presence of mutations in the target gene The real time quantitative PCR qPCR double dye oligonucleotide hydrolysis principle uses specific primers and an internal double dye probe with a reporter and a quencher FAM TAMRA for the amplification reactions In addition a 3 end modified phosphate oligonucleotide is used that perfectly matches the wild type NPM1 gene and does not allow polymerization During PCR if the target of interest is present the probe specifically anneals between the forward and reverse primer sites The DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the
12. genomic DNA Mut PC 300 ul Primers and Probe Mix Total NPM1 PPM Total NPM1 25x 90 ul Primers and Probe Mix Mutated NPM1t PPM Mut NPM1 25x 90 ul Primers and Probe Mix NPM1 MutA PPM NPM1 MutA 25x 90 ul Primers and Probe Mix NPM1 MutB PPM NPM1 MutB 25x 90 ul Primers and Probe Mix NPM1 MutD PPM NPM1 MutD 25x 90 ul Mix of specific reverse and forward primers for total NPMI wild type and mutated plus a specific FAM TAMRA probe t Mix of specific reverse and forward primers including phosphate primer for all mutations of NPM1 plus a specific FAM TAMRA probe Mix of specific reverse and forward primers including phosphate oligonucleotide for the specific detection of NPM1 MutA plus a specific FAM TAMRA probe Mix of specific reverse and forward primers including phosphate oligonucleotide for the specific detection of NPM1 MutB plus a specific FAM TAMRA probe 1 Mix of specific reverse and forward primers including phosphate oligonucleotide for the specific detection of NPM1 MutD plus a specific FAM TAMRA probe Note Vortex and briefly centrifuge the controls and the primers and probe mixes before use 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 6 ipsogen NPM1 MutaScreen Handbook 01 2013 Reagents M Nuclease
13. ipsogen NPM1 MutaScreen Kit at 15 to 30 C and keep primers and probe mixes protected from light See Reagent Storage and Handling page 8 Avoid repeated freezing and thawing b Very low initial amount of target Check the amount of sample DNA En Always check the DNA quality OD469 OD 229 and concentration before starting Note Depending of the chosen method of DNA preparation inhibitory effects may occur Signal absent or low in samples but okay in controls Inhibitory effects of sample Always check the DNA quality material caused by insufficient OD460 OD 29 and concentration purification before starting Repeat DNA preparation Negative H O control is positive Cross contamination reagent Replace all critical reagents contamination instrument error well or capillary inversion or probe degradation Always handle samples kit components and consumables in accordance with commonly accepted practices to prevent carry over contamination Keep primers and probe mixes protected from light Check for false positives on fluorescence curves Check the setup of the reaction ipsogen NPM1 MutaScreen Handbook 01 2013 27 Quality Control This kit is manufactured according to ISO 13485 standard Certificates of analysis are available on request at www qiagen com support References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehe
14. ls provided with this product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties 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 GP BD 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 www qiagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bnI giagen com Brazil suportetecnico brasil giagen com Canada techservice ca qiagen com Chi
15. n controls Mean C values with primers and probe mixes Total Mut NPM1 NPM1 NPM1 Control NPMI NPM1 MutA MutB MutD Water 2 Undetected Undetected Undetected Undetected Undetected negative NPMI WIC 23 84 Undetected Undetected Undetected Undetected NPM1 Mut PC 26 9 30 49 31 88 32 82 33 09 Quality control shows that the run is valid Conditions of rules 1 2 and 3 have been met BE Rule 1 Water control is undetected with all primer and probe mixes M Rule 2 NPM1 WTC is detected with only PPM Total NPM1 M Rule 3 NPM1 Mut PC is detected with all primer and probe mixes Input interval Determination of the input interval using C values of positive controls is shown Input interval Input interval Cy Total npm WTC Cr Total nemi Mut PC 2 3 23 84 26 9 2 3 23 84 29 2 M Rule 4 The input interval for sample validation is determined ipsogen NPM1 MutaScreen Handbook 01 2013 21 Sample validation Sample input validation using the input interval is shown in Table 10 Table 10 Sample input validation Total Sample NPMI 1 26 46 26 12 27 68 28 78 26 36 26 56 25 81 26 89 25 64 oO CO Sa Oo FP OUN Mut NPMI 27 66 32 73 29 12 34 92 27 49 33 73 27 06 27 87 NPMI MutA 28 29 32 78 Undetected Undetected 36 28 Undetected 37 23 38 91 NPM1 MutB Undetected Undetected 29 79 33 57 Undetected Undetected 38 7 Undetected Undetected Unde
16. na techservice cn qiagen com Denmark techservice nordic giagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india giagen com Ireland techservice uk giagen com Italy techservice it qiagen com Japan techservice jp qiagen com Korea South techservice kr giagen com Luxembourg techservice bnl giagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway techservice nordic qiagen com Singapore techservice sg giagen com Sweden techservice nordic qiagen com Switzerland techservice ch giagen com USA techservice us qiagen com QIAGEN UK techservice uk qiagen com ae Sample amp Assay Technologies
17. nalyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories year warranty on parts and labor installation and training not included Rotor Gene Q Real time PCR cycler and High 9001650 5plex HRM System Resolution Melt analyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories l 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 ee EEE ipsogen NPM1 MutaScreen Handbook 01 2013 29 This page intentionally left blank 30 ipsogen NPM1 MutaScreen Handbook 01 2013 This product is intended to be used for life science research only It is not intended for 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 o
18. ncorrect 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 2 Prepare the following qPCR mix for each primers and probe mix according to the number of samples being processed All concentrations are for the final volume of the reaction ipsogen NPM1 MutaScreen Handbook 01 2013 11 Table 2 describes the pipetting scheme for the preparation of one reagent mix calculated to achieve a final reaction volume of 25 pl A pre mix can be prepared according to the number of reactions using the same primer and probe mix PPM Total NPM1 PPM Mut NPM1 PPM NPM1 MutA PPM NPM1 MutB or PPM NPM1 MutD Extra volumes are included to compensate for pipetting error Table 2 Preparation of qPCR mix for PPM Total NPM1 PPM Mut NPM1 PPM NPM1 MutA PPM NPM1 MutB or PPM NPM1 MutD Pre mix 1 reaction 14 1 Final Component ul reactions pl concentration TaqMan Universal PCR Master Mix 2x 22 Wei x Primers and probe mix PPM 25x t 1 gt Nuclease free PCR 65 975 B grade water Sample to be added 50 Sn T at step 4 Total volume 25 0 25 each 3 Dispense 20 ul of the qPCR pre mix per tube 4 Add 5 ul of the material to be quantified 25 ng sample genomic DNA or control in the corresponding tube total volume 25 ul 5 Mix gently by pipe
19. nformation and protocol in this 24 ipsogen NPM1 MutaScreen Handbook 01 2013 handbook or sample and assay technologies for contact information see Contact Information page 28 Comments and suggestions NPM 1 wild type control not detected with PPM Total NPM1 a Pipetting errors or omitted Check pipetting scheme and the setup reagents tube or well inversions of the reaction Repeat the PCR run b Inappropriate storage of kit Aliquot reagents for storage components Store the ipsogen NPM1 MutaScreen Kit at 15 to 30 C and keep primers and probe mixes protected from light See Reagent Storage and Handling page 8 Avoid repeated freezing and thawing NPM1 mutated positive control not detected with PPM Mut NPM1 PPM NPM1 MutA PPM NPM1 MutB or PPM NPM1 MutD a Pipetting errors or omitted Check pipetting scheme and the setup reagents tube or well inversions of the reaction Repeat the PCR run b Inappropriate storage of kit Aliquot reagents for storage components Store the ipsogen NPM1 MutaScreen Kit at 15 to 30 C and keep primers and probe mixes protected from light See Reagent Storage and Handling page 8 Avoid repeated freezing and thawing No signal even in controls a In Rotor Gene Q instruments no Take care to always place a sample to reaction tube in position 1 be tested in position 1 of the rotor b Pipetting errors or omitted Check pipetting scheme and the setup
20. nsive 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 giagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor Symbols The following symbols may appear on the packaging and labeling ake Contains reagents sufficient for lt N gt reactions at Use by Catalog number LO Lot number MA Material number Temperature limitation Manufacturer Consult instructions for use E b k 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 28 ipsogen NPM1 MutaScreen Handbook 01 2013 Ordering Information Product Contents Cat no ipsogen NPM1 For 24 reactions Wild type NPM1 677013 MutaScreen Kit 24 Control Mutated NPM1 Control Primers and Probe Mix PPM Total NPMI Primers and Probe Mix PPM Mutated NPMI Primers and Probe Mix PPM NPM1 MutA Primers and Probe Mix PPM NPM1 MutB Primers and Probe Mix PPM NPM1 MutD Rotor Gene Q for outstanding performance in real time PCR Rotor Gene Q Real time PCR cycler and High 9001580 5plex HRM Platform Resolution Melt a
21. psogen NPM1 MutaScreen Handbook 01 2013 15 Table 5 Preparation of qPCR mix for PPM Total NPM1 PPM Mut NPM1 PPM NPM1 MutA PPM NPM1 MutB or PPM NPM1 MutD Pre mix 1 reaction 14 1 Final Component ul reactions pl concentration TaqMan Universal PCR Master Mix 2x 12 u ir Primers and probe mix PPM 25x 1 gt k Nuclease free PCR 6 5 975 B grade water Sample to be added 50 EN 2 at step 4 Total volume 25 0 25 each 3 Dispense 20 pl of the qPCR pre mix per well 4 Add 5 pl of the material to be quantified 25 ng sample genomic DNA or control in the corresponding well total volume 25 ul 5 Mix gently by pipetting up and down 6 Close the plate and briefly centrifuge 300 x g approximately 10 seconds 7 Place the plate in the thermal cycler according to the manufacturer recommendations 8 Program the thermal cycler with the thermal cycling program and set the instrument for the acquisition of dual labeled FAM fluorescent probe as indicated in Table 6 for Applied Biosystems 7500 and ABI PRISM 7900HT instruments or Table 7 for the LightCycler 480 instrument 16 ipsogen NPM1 MutaScreen Handbook 01 2013 Table 6 Temperature profile for Applied Biosystems 7500 ABI PRISM 7000 ABI PRISM 7700 or ABI PRISM 7900HT instruments Mode of analysis Standard Curve Absolute Quantitation Hold Temperature 50 C Time 2 minutes Hold 2 Temperature 95 C Time 10 minutes Cycling AO times 95 C for 15 seconds
22. ptical density OD of the sample at 260 nm and DNA quality can be determined either by spectrophotometry or gel electrophoresis M The OD y69 OD 9 ratio should be 1 7 1 9 and smaller ratios than this may indicate protein contamination or the presence of organic chemicals M Electrophoretic analysis on a 0 8 1 0 agarose gel should allow the visualization of the isolated DNA as a distinct band of approximately 20 kb a slight smear will give acceptable results The resultant DNA will need to be diluted to a concentration of 5 ng ul in 1x TE buffer at pH 8 0 and then stored at 4 to 8 C for 1 week or at 20 C if longer term storage is required The qPCR reaction is optimized for DNA samples containing 25 ng purified genomic DNA An amount of 250 ng of genomic DNA is needed per sample 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 ipsogen NPM1 MutaScreen Handbook 01 2013 9 Protocol qPCR on Rotor Gene Q 5plex HRM instruments with 72 tube rotor Using this instrument we recommend performing all measurements in duplicate as indicated in Table 1 Table 1 Number of reactions for Rotor Gene Q instruments with 72 tube rotor Samples Reactions With the total NPM1 primers and probe mix PPM Total NPM1 With the mutated NPM1 primers and probe mix PPM Mut NPM1
23. r 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 The purchase of this product allows the purchaser to use it for the performance of diagnostic services for human in vitro diagnostics No general patent or other license of any kind other than this specific right of use from purchase is granted hereby NPM1 mutations and uses thereof are protected by patent rights including European patent applications EP1807448 EP1944316 and EP2319865 US patent 8 222 370 US patent application US2008299560 and foreign counterparts Trademarks QIAGEN HRM ipsogen Rotor Gene QIAGEN Group ABI PRISM Applied Biosystems FAM TAMRA Life Technologies LightCycler TaqMan Roche Group Limited License Agreement for ipsogen NPM1 MutaScreen Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with this product and this 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 any components not included within this Kit except as described in the protoco
24. separate room with specific material pipets tips etc Reagent Storage and Handling The kits are shipped on dry ice and must be stored at 30 C to 15 C upon receipt M Minimize exposure to light of the primers and probe mixes PPM tubes E Gently mix and centrifuge the tubes before opening HE 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 J _ amp _ __ lt lt 8 ipsogen NPM1 MutaScreen Handbook 01 2013 Procedure Sample DNA preparation Genomic DNA should be obtained either from whole blood purified peripheral blood lymphocytes of whole blood polynuclear cells or granulocytes For comparable results it is recommended that the same cellular fraction and DNA extraction method are used DNA extraction can be performed using a commercially available kit or a home brew method DNA quantity should be determined by measuring the o
25. target The probe fragments are then displaced from the target polymerization of the strand continues and a fluorescent signal is released The 3 end of the probe is blocked to prevent extension of the probe during PCR see Figure 1 The phosphate oligonucleotide enhances specificity of the PCR reaction by competition with one PCR primer for a common target site When the PCR template contains the wild type sequence the phosphate oligonucleotide will dominate over PCR primer binding due to higher affinity There is no extension by the DNA polymerase and no amplification is observed When the mutated sequence is present PCR primer binding will dominate over phosphate oligonucleotide binding and amplification will proceed 4 ipsogen NPM1 MutaScreen Handbook 01 2013 PPM Total NPM 1 PPM Mut NPM 1 with 3 P oligo PPM NPM 1 MutA with 3 P oligo Mutated NPM1 gl Non MutA Forward or reverse primer F Double dys probe P Phosphate oligonucleotide 3 P oligo Figure 1 Results obtained with the primers and probe mixes in the ipsogen NPM1 MutaScreen Kit The same principle shown to detect NPM1 MutA applies for NPM1 MutB and NPM1 MutD ipsogen NPM1 MutaScreen Handbook 01 2013 5 Materials Provided Kit contents ipsogen NPM1 MutaScreen Kit 24 Catalog no 677013 Number of reactions 24 NPM1 wild type control wild type NPM1 genomic DNA WTC 300 pl NPM1 mutated positive control NPM1 MutA B D
26. tected Undetected Mean C values with primers and probe mixes NPM1 MutD 38 37 Undetected Undetected Undetected 27 96 33 54 Undetected Undetected Undetected Results of the samples are valid Conditions of Rule 5 have been met E Rule 5 The values of the C Total npmi Sample for all samples are within the input interval 23 84 29 2 NPM1 mutation detection Detection of an NPM1 mutation in the samples is shown in Table 11 ipsogen NPM1 MutaScreen Handbook 01 2013 22 Table 11 NPM1 mutation detection Mean C values with PPM Screening Total Mut Mutation Sample NPM1 NPMI Detected Type 1 26 46 27 66 Yes MutA 2 26 12 32373 Yes MutA 3 27 68 29 12 Yes MutB 4 28 78 34 92 Yes MutB 5 26 36 27 49 Yes MutD 6 26 56 3373 Yes MutD 7 25 81 27 06 Yes Not typed 8 26 89 27 87 Yes Not typed 9 25 64 Undetected No NA Samples positive for NPM1 mutation are detected C Mut npm Sample Conditions of Rule 6 have been met for 8 of 9 samples M Rule 6 A sample is positive for an NPM1 mutation if a Cy value is obtained with PPM Mut NPM1 NPM1 mutation type The procedure used to assign the NPM1 mutation type to samples positive for an NPM1 mutation is shown in Table 12 ipsogen NPM1 MutaScreen Handbook 01 2013 23 Table 12 Typing of NPM1 mutation as MutA MutB or MutD Mean C values with PPM Mut NPMI NPMI NPM1 Sample NPMI MutA Mut B MutD Type 1 27 66 28 29 Undetected 38 37 MutA
27. tting up and down 6 Place the tubes in the thermal cycler according to the manufacturer recommendations 7 Program the Rotor Gene Q instrument with the thermal cycling program as indicated in Table 3 12 ipsogen NPM1 MutaScreen Handbook 01 2013 Table 3 Temperature profile Mode of analysis Quantitation Hold Temperature 50 deg Time 2 mins Hold 2 Temperature 95 deg Time 10 mins Cycling AO times 95 deg for 15 secs 60 deg for 1 min 30 secs with acquisition of FAM fluorescence in channel Green Single 8 For Rotor Gene Q instruments select Slope Correct for the analysis We recommend setting the threshold at 0 03 Start the thermal cycling program as indicated in Table 3 ee EEE ee ipsogen NPM1 MutaScreen Handbook 01 2013 13 Protocol qPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments and LightCycler 480 instrument Using 96 well plate qPCR equipment we recommend performing all measurements in duplicate as indicated in Table 4 Table 4 Number of reactions using 96 well plate qPCR equipment Samples Reactions With the total NPM1 primers and probe mix PPM Total NPM1 With the mutated NPM1 primers and probe mix PPM Mut NPM1 With the NPM1 MutA primers and probe mix PPM NPM1 MutA With the NPM1 MutB primers and probe mix PPM NPM1 MutB With the NPM1 MutD primers and probe mix PPM NPM1 MutD n DNA samples n x 2 reactions 4 reactions wild type control WTC and mut
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