Manual - Trimgen
Contents
1. E 3 Data Analysis Open the analysis software GeneMapper or Genescan Follow the instruction to add the data for analysis The instructions are provided online GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf GeneScan www trimgen com docs PartlV Genescan pdf Confirm results of mutant control M 08 In the sample plot window shows graphic data find the results of the mutant control M 08 Read the peaks from left to right and record peak size in Test Report From download the Form from http Awww trimgen com docs M2 report form GP08 xIs The peak size of the Mutant Controls is the standard for sample analysis Result of M 08 mutant controls Peak Peak Color Peak Size Interpretation 1 Red 34 04 Mutation E542K G1624A 2 Black 35 62 Wildtype 542WT 1624WT 3 Red 41 44 Internal control 4 Blue 44 17 Mutation E545G A1634G 5 Blue 45 06 Wildtype 545WT 1633 WT 6 Black 45 50 Wildtype 545WT 1634 WT 7 Red 46 34 Mutation E545K G1633A 8 Blue 49 83 Mutation H1047R A3140G 9 Red 50 79 Mutation H1047L A3140T 10 Black 52 28 Wildtype 1047WT 3140WT Peak size may vary slightly depending on instrument polymer type and the length of capillary 16 PIK3CA Mutation Analysis Reagents User Manual V1 5 E 4 Data analysis for sample ref http trimgen com docs PI3K data anal2. TrimGen Miutector A Mutation Detection Kit S PIK3CA Mutation Analysis Reagents f os Manual V1 5 f f A Cat No GP08 32 reactions t www trimgen com j PIK3CA Mutation Analysis Reagents User Manual V1 5 Storage Upon receipt of the kit store at 20 C until use At this temperature the reagents are stable for 6 months After first use store all of the reagents at 2 8 C and keep them protected from direct light At this condition the reagents are stable for 1 month For research use only not for use in diagnostic procedures PIK3CA Mutation Analysis Reagents User Manual V1 5 CONTENTS Introduction 5 Overview of Mutector Assay 6 Materials Provided 7 Materials Required 8 Equipment Required 8 DNA Sample Preparation 9 Sequencer Setup 9 Thermal Cycling Programs 10 Mutector Assay Protocol 11 Data Analysis 16 Troubleshooting 18 PIK3CA Mutation Analysis Reagents User Manual V1 5 Notice to Purchaser The Mutector kit is provided as research use only not for use in diagnostic procedures The purchaser must determine the suitability of the product for their particular use TRIMGEN DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL TRIMGEN BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR O
3. 72 C 5 min Hold at 4 C Note y Optional To verify the PCR products run Syl of the PCR products on a 1 2 agarose gel The correct band size is around 100 bp The procedure can be temporarily stopped after Program 1 The PCR product can be stored at 4 C for next day test During the PCR amplification process prepare steps B1 B2 12 PIK3CA Mutation Analysis Reagents User Manual V1 5 PCR Product Clean up B 1 Prepare C UP Mix C Buffer 10 pL x x1 1 of PCR tubes C UP1 1 0 pL x DTA S of PCR tubes C UP2 1 0 pL x x1 1 of PCR tubes Mix the reagents and spin down For pipetting error B 2 Collect 0 2 mL strip tubes one tube for each PCR reaction for example if you have 8 tubes for PCR then collect 8 tubes Label the tubes with C and sample name in the same manner as the PCR tubes B 3 Add 11 uL of C UP mix to each new tube B 4 Transfer 4 uL of each sample s PCR product to corresponding C UP tube B 5 Cap the tubes mix the contents and spin all tubes B 6 Incubate the tubes in a thermal cycler using Program 2 Program 2 37 C for 25 min 95 C for 5 min Hold at 4 C During the product clean up incubation prepare steps C1 C4 13 PIK3CA Mutation Analysis Reagents User Manual V1 5 C STA Mutation Detection C 1 Collect one 2 mL tube and label with ST Mix the ST D reagent and DP 08 detection primer to make the ST Mix The ST Mix ca
4. PIK3CA Mutation Analysis Reagents User Manual V1 5 Mutector Assay Protocol A PCR Amplification Thaw all reagents and keep on ice Spin down the reagents before use Negative and positive controls are recommended to be run each time A 1 Prepare PCR reaction mix PM The PCR reaction mix can be prepared by using the following formula Master Mix 18 x 2 x 1 1 Total of samples PCR P 08 1x 2 x 1 1 Total of samples 2 tubes for negative and positive sample controls Adjustment for pipetting error Collect one 2 mL tube and label with PM PCR reaction mix Transfer the above reagents to the tube and mix the contents This is the master mix for PCR amplification A 2 Collect 0 2 mL PCR strip tubes and label the tubes as follows Sample 1 2 3 200 00 010 Neg Negative control Pos Positive control 11 PIK3CA Mutation Analysis Reagents User Manual V1 5 A 3 Add 19 uL of PM into each of the tubes A 4 Add 1 uL of negative control sample to the Neg tube A 5 Add 1 uL of positive control sample to the Pos tube A 6 Add 1 2 uL of sample DNA 20 80 ng uL to each sample tube When using TrimGen s WaxFree kit for paraffin sample DNA extraction add 1 uL of the final extract to each sample tube A 7 Place the PCR tubes in a thermal cycler and run Program 1 Program 1 1 cycle 94 C 5 min 35 cycles 94 C 30 sec 52 C 30 sec 72 C 30 sec 1 cycle
5. N ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT TRIMGEN IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Limited Product Warranty It is imperative that the users strictly adhere to this manual Failure to do so will void TrimGen s guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose License The purchase of Mutector kit includes a limited nonexclusive license to use the kit This license does not grant rights to reproduce or modify the Mutector kit for resale or to use the Mutector kit to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by estoppels is granted Product Safety and Liabilities When working with the kit reagents always wear a lab coat disposable gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the misuse the results of use or the inability to use this product Trademarks The trademarks mentioned herein are the property of TrimGen or their respective owners TrimGen Corporation All rights reserved I
6. e Loading buffer for ABI capillary type sequencers and special fluorescence labeled size standards PIK3CA Mutation Analysis Reagents User Manual V1 5 Materials Required 0 2 ml PCR tubes 8 well strip tube DS 32 Matrix Standard Kit Applied Biosystems Cat No 4345831 The calibration defines the color definition on the sequencer to read the test results correctly This is a one time set up procedure for all Mutector assays Equipment Required Thermal Cycler Any type of thermal cycler is acceptable for performing the assay Sequencer Any type of Applied Biosystems s Genetic Analyzer or comparable capillary electrophoresis equipment Analysis Software GeneMapper or GeneScan for fragment analysis PIK3CA Mutation Analysis Reagents User Manual V1 5 DNA Sample Preparation Any commercially available DNA extraction kit is acceptable Paraffin FFPE tissue samples A kit specially designed for FFPE sample DNA extraction is available from TrimGen Product information WaxFree DNA for 50 samples Cat WF 50 WaxFree DNA for 100 samples Cat WF 100 DNA concentration When using a column or bead DNA extraction method adjust the final concentration of extracted DNA to 20 80 ng uL When using TrimGen s DNA preparation kit follow the preparation kit protocol to perform the PCR reaction Sequencer Setup First time users should set up the analysis program for the ABI sequencer one time setup A
7. eak If the wild type peak is too high cannot see the top of the peak and the peak is highlighted with pink color your ST reaction is too strong and the small peak may be a pull up from background noise Follow the trouble shooting F2 to dilute the final product of ST reaction with de ionized formamide After dilution reload the sample If you can see the top of the wild type peak then use the following calculation to identify the small peak Ratio Height of mutant peak Height of wild type peak If the ratio is larger than 0 08 the peak is considered to be a mutation peak this ratio does not represent the percentage of the mutation present in the sample Otherwise the peak is background pull up and does not indicate the presence of a mutation in the sample 20
8. fter correction of size standard 18 PIK3CA Mutation Analysis Reagents User Manual V1 5 F 6 F 7 F 8 Check the raw data if the signals from the sample and size standard are too low the capillary tube may be blocked by a bubble The sample needs to be re loaded If the sample signal is too strong and the size standard is too low the STA products will compete with the size standard DNA to enter the capillary tube Diluting the final STA product with de ionized water and reloading the sample will easily resolve this problem Graphic data does not automatically appear Check the raw data if the signals from the sample and size standard are too low the capillary tube is blocked by a bubble The sample needs to be re loaded When adding a sample to the loading plate do not use the pipettor to mix the sample as this will generate bubbles The sample DNA in the loading plate will automatically enter into the capillary tube by electric current If the sample signal is too strong and the size standard is too low the ST products will compete with the size standard DNA to enter the capillary tube When the size standard is too low the software can not detect the size standard correctly and the program will not show the graphic data Diluting the final ST product with de ionized water and reloading the sample will easily resolve this problem The size standard may have miscalculated Check the size standard and manual
9. fter setup the program can perform data analysis for all Mutector II tests Please choose either GeneMapper or GeneScan to analyze your data GeneMapper Analysis Step I GeneMapper Setup www trimgen com docs Partl GeneMapper Setup pdf Step Il Data Collection Software Setup www trimgen com docs Partll Data Collection Setup pdf Step Ill Data Analysis Using GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf GeneScan Analysis Step I Data Collection Software Setup www trimgen com docs Partll Data Collection Setup pdf Step Il GeneScan Setup and Data Analysis www trimgen com docs PartlV Genescan pdf PIK3CA Mutation Analysis Reagents User Manual V1 5 an Important Spectral calibration is required before running the test To read the test results correctly the sequencer needs to be calibrated with the DS 32 calibration kit Applied Biosystems Cat No 4345831 This is a one time calibration to set up the correct spectral channels Refer to the DS 32 Matrix standards kit manual to perform spectral calibration Thermal Cycling Programs Program 1 PCR amplification 1 cycle 94 C 5 min 35 cycles 94 C 30 sec 52 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min Hold at 4 C Program 2 C UP treatment 37 C 25 min 95 C 5 min Hold at 4 C Program 3 STA reaction 1 cycle 94 C 4 minute 20 cycles 94 C 20 sec 55 C 30 sec 70 C 20 sec Hold at 4 C
10. ly correct the size standard see the sequencer s instruction manual Reanalyze the data after correction of size standard Missing wild type peak s The wild type peaks are built in reaction controls for sample DNA amplification these peaks should appear in all samples If wild type peaks are not observed it indicates that the PCR amplification failed The possible causes could be poor DNA quality low DNA concentration or PCR inhibitor in the DNA sample Background noise The background of the assay is normally low When the STA reaction is too strong peak signal over 8000 rfu and highlighted 19 PIK3CA Mutation Analysis Reagents User Manual V1 5 F 8 F 9 with pink color background noise also called pull up can be seen To resolve this issue simply dilute the final ST product with de ionized water and re load the sample Overloading DNA is another cause of increased background noise Reducing the input DNA amount will resolve this issue Unexpected peak Presence of a peak that does not match any of the peaks in the Mutation Control The Mutation Control contains 13 peaks wild type and mutant peaks If you see a peak that does not match the Mutation Control please contact Technical Support for further analysis Mutation peak cut off For some samples there could be a small peak observed in one of the mutation positions To verify the peak you need to confirm the signal strength of the wild type p
11. n be prepared by using the following formula ST Mix ST D 11 x 1 x 1 1 Total of C UP samples DP 08 2 x 1 x 1 1 Total of C UP samples One extra tube for M 08 Mutant Controls Adjustment for pipetting error Add the reagents to the tube and mix gently C 2 Collect 0 2 ml strip tubes one tube for each C UP treated sample Add one new tube for M 08 Mutant Controls and label the tube with CTL Sample 1 2 3 New tube for M 08 mutant controls AD The M 08 mutant controls is designed for data analysis ZA and must be run each time C 3 Add 13 uL of ST Mix into each tube C 4 Transfer 2 pL of C UP treated PCR product to each corresponding tube C 5 Add 2 uL of M 08 into the CTL tube C 6 Cap the tubes mix the contents and spin down all tubes C 7 Place the tubes into the thermal cycler and perform the ST reaction using Program 3 14 PIK3CA Mutation Analysis Reagents User Manual V1 5 Program 3 1 cycle 94 C 4 min 20 cycles 94 C 20 sec 55 C 30 sec 70 C 20 sec Hold at 4 C Sample Loading D 1 Add 15 uL of the Loading Buffer to each well of a sequencer adapter plate D 2 Transfer 5 uL of the ST products into each well Confirm and remove any bubbles in the well D 3 Load the plate into the sequencer and run the preset Data Collection Program GeneMapper or GeneScan 15 PIK3CA Mutation Analysis Reagents User Manual V1 5 E E 1 E 2
12. nformation in this document is subject to change without notice PIK3CA Mutation Analysis Reagents User Manual V1 5 Introduction Phosphoinositide 3 kinase PI3K plays an important role in the EGFR signaling pathway The PI3K activates the serine threonine kinase AKT which in turn regulates several signaling pathways controlling cell survival growth and proliferation The gene PIK3CA encoding the catalytic subunit of the enzyme has been found to mutate frequently in human cancers The studies found that the PISKCA mutations in cancer cells associated with early recurrence poor prognosis and drug resistance The Mutector PIK3CA Mutation Analysis Reagents uses TrimGen s proprietary technology called Shifted Termination Assay STA to detect the following 5 mutations E542K G1624A exon 9 E545K G1633A exon 9 E545G A1634G exon 9 H1047R A3140G exon 20 H1047L A3140T exon 20 The kit contains reagents enough for 32 reactions Shifted Termination Assay STA Shifted Termination Assay is a proprietary multi base primer extension method STA technology extends primers by multiple bases to increase signal strength and fragment size creating mutation peaks that are easily distinguished from wild type Mutations are confirmed by both peak color and size The STA technology can detect low level mutations often missed by sequencing Mutant O Wild type gt X Mutant Wild type J j gt aX Fragme
13. nt analysis STA reaction PIK3CA Mutation Analysis Reagents Overview of Mutector Assay User Manual V1 5 Total assay time 2 3 hours hands on time 10 20 min Load sample to sequencer Wild type Mutation PCR amplification 1 5 hours Time varies depending on the type of thermal cycler used PCR product clean up 30 min Mutation detection ST reaction 40 min Time varies depending on the type of thermal cycler used 5 min Capillary Electrophoresis Fragment analysis 30 40 min Time varies depending on the type of sequencer PIK3CA Mutation Analysis Reagents User Manual V1 5 Materials Provided The Mutector PI3K Mutation Analysis Reagents can perform 32 reactions Materials Quantity Master Mix 700 pL PCR P 08 50 uL C UP1 40 uL C UP2 40 uL C UP Buffer 430 pL ST D 430 pL DP 08 80 pL M 08 50 uL Loading Bfx 1000 pL Light sensitive Keep these reagents protected from direct light Reagent Description Master Mix Reagents for DNA amplification PCR P 08 PCR primer mix for amplification of PIK3CA gene C UP1 and C UP2 Enzymes for cleanup of PCR products C UP Buffer Buffer for C UP1 and C UP2 ST D Light sensitive Pre mixed STA reagents for mutation detection DP 08 Detection primers for PIK3CA gene M 08 Mutant and wild type control DNA for PIK3CA gene The controls are sufficient for 32 test runs Loading Bfx Light sensitiv
14. sidered mutation peaks The size standard is too high The height of the size standard varies with STA reaction efficiency Generally the size standard intensity green color is 2 000 3 000 rfu When the STA efficiency is too low poor DNA quality improper handling of STA reagents the size standard may reach 5 000 7 000 rfu Diluting the size standard with de ionized formamide will reduce the size standard signal The signal peak is too high The assay chemistry is optimized to detect mutations in samples with low DNA concentration such as DNA from a fine needle aspiration FNA sample For regular FFPE samples the assay signal may be too high to analyze peak height gt 8000 the top of the peak is not visible or the peak is highlighted with pink color Diluting the final STA product with de ionized water can effectively reduce the signal Do not dilute the assay reagents as it will cause improper enzyme reactions and generate false results Each laboratory has different PCR instruments and the signal strength may vary among the laboratories The first time user should define the dilution factor 2 20 times dilution during initial validation Once the dilution factor is determined most samples will have a consistent result Data failed to be analyzed The size standard may be miscalculated by the sequencer Check the size standard and manually correct it see the sequencer s instruction manual Reanalyze the data a
15. ysis pdf Four wild type peaks are observed in every sample If all four peaks are not observed it indicates that the DNA amplification failed see troubleshooting section or the sample is 100 mutation s such as mutant cell lines If sample contains mutant DNA the mutation s will show as additional peak s Compare the peak size and color with the mutant controls M 08 The peak size may be slightly shifted due to migration differences between individual capillary tubes Comparing the wild type peak of the sample with the wild type peak of the Mutant Controls can identify the migration shift Fill in the peak size to the Test Report From Any peak that does not match the Mutant Controls will not be considered as evidence of a mutation Example of assay result Sample FFPE sample one section 1 x 0 5 cm 10 um DNA extraction WaxFree DNA kit H1047R Wild type Wild type A3140G 2 peaks Blue peak Wild type 17 PIK3CA Mutation Analysis Reagents User Manual V1 5 F Troubleshooting F 1 F 2 F 3 F 4 Color leak through When the sample DNA concentration is too high the STA reaction generates a strong fluorescent signal gt 5 000 rfu Fluorescence spillover into other channels will occur for example the black peak of the wild type signal may be observed in the red and or blue channels The leak through peaks are the same size as the original peak and should not be con
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