ICEme Kit User Manual
Contents
1. ICEme MX ICP Mutation Enrichment Kit User Guide for the ICEme Kit Multiplex ICE COLD PCR Mutation Enrichment Kit Transgenomic Advancing Personalized Medicine This product is for Research Use Only Table of Contents Reagent PrepalraliGNo 606seeceser sid ose nd see Ket ete AE TA EE ENEON 2 TAOS oe ooo uce oe ce ee de es ee ebc bee E eee oats auntie 2 Principles of the ICEme Kit for Mutation Detection 0 ce eee eee ee 2 Analysis of Samples using ICEME Kit 22625 28cs0csshancesccedinabeicsesansisencauesans 2 Kit Components os soc w ten cacicdinseenegaaddesodeas siaenbkeedusadeageeurda 3 MX PCR and MX ICP Kit Components ccc avinsdsie cas eauesesasnareiaiduse tind sddea yews 4 Number of Samples that can be tested with one ICEme Kit 0 ee eee eee eee 4 Additional Required Equipment and Reagents cece ee ee eee eeee 4 Primary Sample Collection Handling and Storage eee ee ee eee 4 MX PCR Step by Step Instructions 0 cece cece eee eee rene 4 MX PCR Pre Amplification Protocol vanses cscs vo shecoede bes dnee soa eSen eee deus on eeeee ss 5 Thermal Cycler Program for the MX PCR Amplification Protocol 2 008 6 Quality Control of MX PCR Products optional c lt lt sics42cese0ussa0ce00beadeicaweuesedis 6 MX PCR Product ANalySi Sessi pws esd earn Gane o eeu eee aout down shee pe PONET 4 eee ees 7 Ueto Positive CONWOL ccs chested seas2. EGFR Exons 12 18 19 20 and 21 Subsequent sequence analysis of MX ICP products is performed on any downstream sequence analysis platform currently in use MX PCR products produced using this kit range from 130 220 bp MX ICP products vary in length but they are all less than 200 bp MX ICP products can be used as templates for the user selected particular downstream sequence detection platform Note This is a Research Use Only kit Sale of this product is conditioned on the Limited Use License which you accept by purchasing this product and is available for download from within the Documentation Tab on ICEme Kits Details and Ordering Page on the Iransgenomic website Reagent Preparation All reagents supplied with this ICEme Kit are ready to use Some components will need to be thawed vortexed and or spun in a microcentrifuge before use check details in Assay Procedure below Reagents do need to be combined to produce Master Mixes and reaction mixtures full details are given in the Assay Procedure below Storage The kit should be stored between 18 C and 25 C in a constant temperature freezer until use Principles of the ICEme Kit for Mutation Detection This kit uses Transgenomic s proprietary primer sets for MX PCR amplification and MX ICP mutation enrichment A general overview of the process is shown in Overview of Multiplexed ICE COLD PCR MX ICP located within the Documentation Tab on ICEme Kits Details and Ord
3. ICEme Kit D Use Table 5 as a guide for preparing the Master Mix for each MX ICP reaction assuming 1 0 uL of 1 200 or 1 10 for KRAS 4B dilution of MX PCR product is to be used reaction Table 5 Master Mix Preparation Volumes per Reaction Phusion Polymerase Number of Reactions Sas KRAS Exon 2 amp BRAF Exon 15 ONLY Volume of Water 36 0 uL 31 375 uL Polymerase Buffer 5 0 uL 10 0 uL dNTPs 4 0 uL 4 0 uL MX ICP Primers 1 0 uL 1 0 uL RS oligo 2 5 uL 2 5 uL DNA Polymerase 0 5 uL 0 125 uL Total Volume Master Mix 49 0 uL 49 0 uL E Calculate required volumes for any given Master Mix by reference to the chart above Note an additional reaction will be required for a No Template Control NTC2 Note take into consideration that a Master Mix volume slightly greater than this calculation will be required to allow for losses during pipetting Label 0 2 mL PCR tubes or wells of a 96 well plate with appropriate sample information Label a 1 7 mL centrifuge tube for Master Mix preparation H Add required volumes of molecular biology grade water Polymerase Buffer dNTPs MX ICP Primers and RS oligo to Master Mix tube Take the DNA Polymerase tube out of the freezer vortex for 10 sec briefly centrifuge and add required volume to Master Mix tube J Cap Master Mix tube vortex for 10 sec briefly centrifuge and store on ice until use K Pipette 49 0 uL of Master Mix into appropriate wells changing pipette tips in between if
4. corresponding to the main MX PCR product should be observed 4 If no product is observed ensure quality of input template DNA was sufficient 5 No MX PCR products should be visible in the NTC sample A If DNA products are visible with NTC contamination is likely and the sample should not be taken forward 6 Use your laboratory s procedures for NGS analysis of the test samples TIP AT THIS STAGE MX PCR PRODUCTS CAN BE STORED AT LESS THAN OR EQUAL TO 20 C PRIOR TO ENRICHMENT WITH MX ICP User Guide 6 ICEme Kit MX PCR Product Analysis The PCR product generated by MX PCR pre amplification reactions can be used in any of the kit s MX ICP assay s Use of Positive Control See the document Positive Control for ICEme Kit located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Limitations of the MX PCR Assay Procedure See the document Limitations of ICEme Kit located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website MX ICP Step by Step Instructions Please refer to Template Considerations before using this protocol 1 Procedure Setup Ensure that the following ramp rates are used depending on thermal cycler A C1000 1 5 C sec B Veriti 38 5 C Tetrad default 3 0 C sec 2 Preparation of Template DNA for ICE COLD PCR Analysis Template DNA will contain amplified product from 15 amplicons A Qubit analysis should be perfo
5. the tumor area from the glass slide using a fresh sterile scalpel for each new slide It is recommended that at least two independent analyses are performed for each sample For successful use of this kit the extracted DNA should meet the criteria listed in the document Template Considerations for Extracted DNA located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Note 1 Extracted DNA samples not intended for immediate analysis with this kit should be stored frozen at 20 C to 80 C 2 The PCR product from the MX PCR pre amplification reaction can be stored frozen at 20 C to 80 C prior to use in MX ICP assays 3 The PCR product following the MX ICP enrichment assay can be stored frozen at 20 C to 80 C prior to use in the downstream sequence detection platform used by the laboratory 4 Exposure of any frozen sample to repeated freeze thaw cycles should be avoided MX PCR Step by Step Instructions Please refer to the document Jemplate Considerations for Extracted DNA located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website before using this protocol User Guide ICEme Kit MX PCR Pre Amplification Protocol il 2 Remove MX PCR Primer Mix dNTPs and 5X GC Buffer from freezer and thaw on ice Once thawed vortex all kit components 10 seconds to mix thoroughly Briefly centrifuge 10 seconds to ensure no liquid re
6. well plate with appropriate sample information Label a 1 7 mL centrifuge tube for MX PCR Master Mix preparation Add required volume of UV treated molecular biology grade water to 1 7 mL centrifuge tube labeled MX PCR Master Mix Add required amount of 5X GC Buffer dNTPs and MX PCR Primer Mix to MX PCR Master Mix tubes Take the DNA Polymerase out of the freezer vortex for 10 seconds centrifuge for 10 seconds and add required volume of DNA Polymerase to the MX PCR Master Mix tube Cap MX PCR Master Mix tube Before use vortex MX PCR Master Mix tube for 30 seconds and centrifuge for 10 seconds Store on ice until use Pipette 35 0 uL MX PCR Master Mix into appropriate wells changing pipette tips in between uses if using a single channel pipettor If using a repeat pipettor ensure that there is no spillage or splashing from well to well Keep the plate on ice User Guide ICEme Kit 14 To appropriate wells add 15 0 uL of each sample template DNA or water No Template Control NTC Use separate pipette tips for each sample and avoid cross contamination of the samples by splashing Cap wells containing sample DNAs and NTC with 8 cap strips if using a 96 well plate or cap 0 2 mL PCR tubes Make sure caps are sealed securely Note Good practice is to place No Template Controls NTC in wells that are not adjacent to wells containing Positive Controls or Test Samples 15 Optional sample only after Step 14 is com
7. with the kit s components depends upon the average batch size tested at any one time because at least one No Template Control NTC must be run for each batch Additional Required Equipment and Reagents For additional components and equipment required to use the ICEme Kit see the document Reagents and Equipment required for ICEme Kit located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Primary Sample Collection Handling and Storage The ICEme Kit can be used with the following e DNA extracted from formalin fixed paraffin embedded tumor samples FFPE slides amp blocks or fine needle aspirations FNAs e Circulating free DNA cfDNA from plasma or serum e DNA isolated from other body fluids For optimal DNA extraction from FFPE the tissue should be fixed in neutral buffered formalin for 14 24 hours placed in ethanol and then embedded in paraffin following standard histological practices Tumor biopsies are a heterogeneous mixture of tumor cells and non tumor cells In addition the tumor itself is a heterogeneous mixture of tumor cells with mutations and tumor cells without mutations Because these somatic mutations may not be evenly distributed throughout the tumor the resultant mutational analysis of different sections from the same tumor may be different To increase the probability of detecting a mutation DNA from the tumor region of the tissue should be isolated by scraping only
8. NRAS 2 NRAS 3 EX 11 EX 15 Initial o i Denaturing ki sole aes Table 6b Thermal Cycler Protocols for MX ICP Amplification 5 cycles Amplification Final Extension 5 min Temperature MX ICP EGFR EGFR EGFR EGFR EGFR PIK3CA PIK3CA Reaction Ex 12 Ex 18 Ex 19 Ex 20 Ex 21 Ex 9 Ex 20 Initial o Denaturing 30 cycles User Guide 9 ICEme Kit Amplification 5 cycles Amplification Final Extension Note Store the samples at 20 C until sequence analysis Quality Control of MX ICP Products optional 1 Analyze an aliquot of MX ICP product with an aliquot of 100 bp DNA mass ladder on a 2 agarose gel to estimate to estimate amplified DNA concentration Only a single band corresponding to the main MX ICP product should be observed If multiple bands are present check quality of input DNA If no product is observed ensure quality of input template DNA was sufficient ot a yk No MX ICP products should be visible in No Template Control sample If DNA products are visible with this control contamination is likely and sample should not be taken to sequence analysis 6 Using your laboratory s procedures proceed to sequence analysis of the test samples MX ICP Product Analysis The PCR product generated by the MX ICP enrichment reactions can now be used for DNA sequence analysis using standard laboratory procedures for the particular platform chosen I
9. ated within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Kit Components The ICEme Kit contains the components needed to perform MX PCR pre amplification and MX ICP mutation enrichment Table 1 MX PCR Kit Components MX PCR Kit Components Tube Cap Color No Tubes in Kit Volume 8 reactions 24 Reactions Phusion HS II DNA Polymerase Orange 1 4 uL 12 uL 5X GC Buffer for Phusion HS II White 1 1 5 mL 1 5 mL dNTPs 10mM Clear 1 55 uL 125 ul MX PCR Primer Mix Blue 1 20 uL 60 uL MX ICP Positive Control Black 1 15 uL 15 uL dNTPs 10mM 2 5 mM each of dATP dCTP dGTP and TTP Table 2 MX ICP Kit Components MX PCR Kit Components Tube Cap Color No Tubes in Kit 8 reactions 24 Reactions Phusion HS II DNA Polymerase Orange 1 4uL 12 uL JumpStart Taq DNA Polymerase Red 1 12 uL 12 uL 5X GC Buffer for Phusion HS II White 1 1 5 mL 1 5 mL 10X JumpStart Reaction Buffer White 1 150 uL 150 uL dNTPs 10mM Clear 1 55 uL 125 ul MX ICP Primer Green 1 8 uL 24 uL RS oligo Yellow 1 20 uL 60 uL dNTPs 10mM 2 5 mM each of dATP dCTP dGTP and TTP User Guide ICEme Kit MX PCR and MX ICP Kit Components There is an ICEme Kit available for each exon The polymerase supplied with a specific exon kit is indicated in the section entitled Thermal Cycler Program for MX ICP Enrichment Protocol Number of Samples that can be tested with one ICEme Kit The total number of samples that can be tested
10. eagents pipetted from tubes in this kit are intended for the setup of Master Mixes and should not be stored for the setup of subsequent analyses User Guide ICEme Kit Resources For additional product information such as troubleshooting guides SDS documentation frequently asked questions Limited Use License or ordering details please visit the Transgenomic website transgenomic com iceme Quality Statement ICEme Kit demonstrates enrichment of mutant DNA sequences in excess of Wild Type DNA through selective amplification of mutant DNA using kit control template and reagents Licenses Trademarks amp Copyright DNA Polymerase Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 789 224 5 618 711 6 127 155 and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as the patented 5 Nuclease Process claims in US Patents Nos 5 210 015 and 5 487 972 no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by e
11. ering Page on the Transgenomic website It is recommended that laboratory set up is optimized to avoid sample or control cross contamination For an example of an ideal laboratory set up see Laboratory Setup to perform Multiplexed ICE COLD PCR MX ICP located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Analysis of Samples using ICEme Kit The ICEme Kit should only be used in the context of the workflow indicated in Figure 1 All components necessary for downstream sequencing platforms must be supplied by the laboratory using this kit Figure 1 ICEme Kit workflow for Mutation Analysis any Downstream Sequencing Platform Patient Downstream Sample Sequence Detection Platform User Guide ICEme Kit Note 1 Due to heterogeneity associated with tumors biopsy samples may contain normal cells as well as Wild Type and mutant tumor cells 2 The Limit of Detection LOD of any mutations present in the sample DNA following MX PCR is dependent on the sensitivity of the downstream sequence detection platform used 3 Only the DNA Polymerases supplied with this kit should be used as indicated for the MX PCR and MX ICP assays 4 Please follow the specific instructions for your laboratory s downstream sequence detection platform by consulting the appropriate instruction manual First time users should perform control experiments outlined in the document Positive Control for ICEme Kit loc
12. f your downstream platform of choice is SANGER then please use the sequencing primer one direction only in Table 7 for your analysis Table 7 M13 Sanger Sequencing Primer M13seq R AGGAAACAGCTATGACCAT If your downstream platform of choice is NGS then please see the amplicon targeted regions for MX PCR and MX ICP Use of Positive Control See the document Positive Control for ICEme Kit located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Limitations of the MX ICP Assay Procedure See the document Limitations of ICEme Kit located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website Warnings amp Precautions None of the reagents in this kit present a health hazard in the quantities supplied Transgenomic s document number SDS 71600X can be found in the document ICEme Kit SDS located within the Documentation Tab on ICEme Kits Details and Ordering Page on the Transgenomic website There are no substances in this kit of animal or human origin that present a risk of infection This kit should be used only by those persons who have been trained in the appropriate laboratory techniques When working with the components of this kit always wear a suitable lab coat disposable gloves and eye protection After use the kit components should be disposed of as clinical waste and in accordance with your local rules and regulations Aliquots of r
13. mains on tube lids and place on ice Prepare MX PCR Master Mix on ice Table 3 MX PCR Master Mix Guidance for 10 ng pL DNA samples MX PCR Master Mix 1X Volume Calculation UV treated Molecular Biology Grade Water uL 18 5X GC Buffer uL 10 dNTPs pL 4 MX PCR Primer Mix uL 2 5 Phusion HS II DNA Polymerase pL 0 5 Total Volume MX PCR Master Mix for 1 reaction uL 35 0 Volume DNA added to reaction 150 ng total 15 0 Multiply volumes in this table by the number of samples being tested Use Table 3 as a guide for preparing the MX PCR Master Mix for MX PCR reactions Amount of UV treated Molecular Biology Grade Water and volume of DNA can be adjusted accordingly 150 ng of DNA should be used for each MX PCR reaction in order to potentially detect gt 0 01 mutation present in the starting material Up to 33 uL DNA can be used in each MX PCR reaction with adjustment of volume of water used Note If DNA contains contaminants ethanol carryover EDTA etc increasing DNA volume may decrease MX PCR efficiency Calculate required volumes for any given MX PCR Master Mix referencing Table 3 If required a Positive Control Mixture may be used in place of a sample there is enough positive control for 3 reactions Note Take into consideration that an MX PCR Master Mix volume slightly greater than the calculation above will be required to allow for losses during pipetting 7 8 9 Label 0 2 mL PCR tubes or wells of a 96
14. on eee unan erase ates dese teaoraneunee epatenscaues 7 Limitations of the MX PCR Assay Procedure 2 cece eee cece eee eee ee ence 7 MA ICP Step by Step INStPUCHONS 3 lt evscccanasteedeivs di edeievadeusseus sees 7 Thermal Cycler Program for MX ICP Enrichment 0 eee ee eee eee eee e eens 9 Quality Control of MX ICP Products optional 2 lt 200 lt ssec0e esses eesseceesauesawees 10 MX ICP Product ANAIySIS i o2546 wtcapeeeeeleweee dae trt cheer eed oS ESee eens eeeeuied 10 Use OF Positive Control co v ceosoyees deen dchus UON UNS VTO IEOU UNUDUN ERUTEN EET 10 Limitations of the MX ICP Assay Procedure 2 eee eee cece eee eee eeeeeeee 10 Warnings amp Precautions 6 25 dceuec tence bccc cu duewed oeere ene tuseuewed dn ee tess 10 RESOULCES erro cscs ree tesyorsnte carers cece ton sae oes mere ere eck esuae we 11 Quality Statement s1 212050 sensencecesennebeasetas teense EE sted ed A AE 11 Licenses Trademarks amp Copyright 2 cece eee eee eee eens 11 User Guide ICEme Kit PLEASE READ THIS USER GUIDE THOROUGHLY BEFORE YOU START USING THIS PRODUCT THIS USER GUIDE CAN BE DOWNLOADED FROM THE TRANSGENOMIC WEBSITE Transgenomic s ICEme Kit is an in vitro assay that uses multiplexed PCR MX PCR followed by multiplexed ICE COLD PCR MX ICP for amplification and enrichment of mutations in KRAS Exons 2 3 and 4 NRAS Exons 2 3 and 4 BRAF Exons 11 and 15 PIK3CA Exons 9 and 20
15. plete open MX ICP Positive Control Pipette an additional 10 uL of Molecular Biology Grade H O and 5 uL of MX ICP Positive Control into Positive Control well tube and cap well tube Addition of the kit positive control as the last sample to be added lessens the chance of contaminating any test sample DNA Cap each well with 8 cap strips if using a 96 well plate or cap 0 2 mL PCR tubes Make sure caps are sealed securely 16 Vortex 1 2 speed for 10 seconds Centrifuge for 20 seconds to ensure all solutions are collected at the bottom of wells or tubes Verify that solutions are at the bottom of each well or tube If not repeat centrifugation Thermal Cycler Program for the MX PCR Amplification Protocol 1 Use the thermal cycler protocol in Table 4 for MX PCR Amplification Ramp Rates C1000 1 5 C sec Veriti 38 5 Tetrad default 3 0 C sec Table 4 MX PCR Amplification Thermal Cycler Protocol Cycles Temp C Time Initial Denaturation 1 98 C 30 sec 15 98 C 10 sec Peah 15 62 C 0 5 C cycle 20 sec 15 72 C 20 sec 20 98 C 10 sec Amplification 20 55 C 20 sec 20 72 C 20 sec Final Extension 1 72 C 5 min Hold 12 C Hold Note Store samples at 20 C Quality Control of MX PCR Products optional 1 Analyze MX PCR product aliquots with an aliquot of a standard 100 bp DNA mass ladder on a 2 agarose gel 2 Use the ladder to estimate MX PCR amplified DNA concentration 3 Bands ranging from 130 220 bp
16. rmed on the MX PCR product For those samples and controls with Qubit values gt 7 ng uL dilute the MX PCR product 1 200 in water except KRAS 4B which would be 1 10 in water Il Dilute the No Template Control from the MX PCR plate NTC1 1 200 in water Ill For those samples with Qubit values lt 7 ng uL DO NOT PROCEED with MX ICP Analysis a The MX PCR should be repeated with more starting DNA b An additional sample may be needed B If previously prepared and stored frozen allow to thaw prior to use 3 MX ICP after MX PCR A The following procedures should be performed in PCR Workstations Use appropriate pipettes to aliquot reagents Il Prior to PCR setup a Turn on UV crosslinker and allow to warm up equilibrate for 5 min b Prior to preparing Master Mixes UV crosslink all empty Master Mix tubes Also UV crosslink 1 7 mL tubes containing appropriate volume of Molecular Biology Grade Water needed for Master Mix preparation These tubes should be UV irradiated for 10 min 600 sec c Make sure all work areas are prepared for analysis of low level mutations This includes correct use of the PCR Workstation dedicated pipettes tips 10 bleach solution and DNA Away solutions B Remove MX ICP Primers dNTPs RS oligo and Polymerase Buffer tubes from freezer thaw on ice vortex each for 10 sec and briefly centrifuge tubes to ensure no sample is on tube cap or sides of the tubes C Prepare Master Mix on ice User Guide
17. stoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Transgenomic Advancing Personalized Medicine For more information visit transgenomic com iceme Transgenomic Inc 12325 Emmet Street Omaha NE 68164 USA 888 813 7253 info transgenomic com transgenomic com Transgenomic is a registered trademark ICEme the helix and MX ICP logos are trademarks of Transgenomic Inc All other trademarks are trademarks of their respective holders 2015 Transgenomic Inc All rights reserved Document No 602435 00 06 28 15 User Guide ICEme Kit
18. using a single channel pipette If using a repeat pipettor ensure that there is no spillage splashing from well to well Keep plate tubes on ice L Vortex and spin down diluted MX PCR product and No Template Control M To appropriate well add 1 0 uL water to MX ICP no template control NTC2 N To appropriate well add 1 0 uL diluted No Template Control from MX PCR NTC1 Note Cap no template controls before adding samples from MX PCR products this includes samples reference controls and cell line positive control O To appropriate well add 1 0 uL diluted sample from MX PCR TU To appropriate well add 1 0 uL diluted mutation positive control from MX PCR if applicable Q Once pipetting is complete cap each column with cap strips if using a 96 well plate or cap 0 2 mL PCR tubes Make sure caps are sealed securely R Vortex 1 2 speed for 10 seconds Centrifuge for 20 seconds t o ensure all solutions are collected at the bottom of wells or tubes Verify that solutions are at the bottom of each well or tube If not repeat centrifugation User Guide 8 ICEme Kit Thermal Cycler Program for MX ICP Enrichment Use the thermal cycler protocols in Tables 6a and 6b for MX ICP Enrichment For specific ramp rates for thermal cycler see Step 1 Procedure Setup under the section MX ICP Step by Step Instructions Table 6a Thermal Cycler Protocols for MX ICP Temperature MX ICP a ae vie ee BRAF BRAF Reston KRAS 2 KRAS 3
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