AmoyDx TM PIK3CA Five Mutations Detection Kit
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1 AmoyDx TM PIK3CA Five Mutations Detection Kit Detection of five mutations in PIK3CA gene Instructions For Use Instructions Version: B1.3 Date of Revision: April 2012 Store at -20±2 1/5
2 Background Phosphatidylinositol 3-kinases (PI3Ks), are a group of enzymes, involved in a diverse range of cellular functions including cell proliferation, differentiation, survival and intracellular trafficking. The product of the phosphoinositide-3-kinase catalytic alpha (PIK3CA) gene is a downstream signal transducer of the epidermal growth factor receptor (EGFR). PIK3CA is somatically mutated in several types of human cancer, such as colorectal cancers (CRC), breast cancer and lung cancer. Activating mutations of the PIK3CA gene are associated with drug resistance to EGFR-TKIs through persistent activation of PI3K/Akt pathway. Intended Use The AmoyDx TM PIK3CA Five Mutations Detection Kit is highly selective and sensitive in detection of five mutations in the PIK3CA gene (Table 1). AmoyDx s patented technology allows detection of 1-2% mutant DNA in a background of 99-98% normal DNA, while ensuring that false negatives are minimized. The AmoyDx TM PIK3CA Five Mutations Detection Kit is intended for research use only. Table 1. Details of five somatic mutations in PCK3CA gene Name Mutation Base Change Cosmic ID PI-M1 H1047R CAT>CGT 775 PI-M2 H1047L CAT>CTT 776 PI-M3 E542K GAA>AAA 760 PI-M4 E545K GAG> AAG 763 PI-M5 E545D GAG> GAT 765 Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 2). The assay uses six wells per sample. Table 2. Kit Contents Tube Reagents Supplied Volume Channel 1 H1047R Reaction Mixture 650 µl 2 H1047L Reaction Mixture 650 µl 3 E542K Reaction Mixture 650 µl 4 E545K Reaction Mixture 650 µl 5 E545D Reaction Mixture 650 µl 6 External Control Reaction Mixture 650 µl FAM 7 PIK3CA Taq DNA Polymerase 50 µl / 8 PIK3CA Mixed Standard 250 µl / Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, LightCycler480Ⅰ and Ⅱ. 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filtered pipette tips for handling DNA samples. 4. Sterile, nuclease-free H2O. 2/5
3 Shipping and Storage The kit requires cold-chain-transportation and be stored immediately upon receipt at -20±2 o Cina constant-temperature freezer and protected from light. The shelf-life of the kit is indicated on the package. Specimen Material Human genomic DNA must be extracted from tissue, blood or fixed paraffin-embedded tissue prior to use and stored at -20±2. Good DNA quality is essential and we recommend use of Qiagen DNA extraction kit (QIAamp DNA FFPE Tissue Kit, cat No , for paraffin embedded specimens; DNeasy Blood & Tissue kit, cat. No or 69506, for tissue and blood specimens). The OD value of DNA samples should be measured using the spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended. Make sure A260/A230 value is greater than 2.0 and A260/A280 value between 1.8 and 2.0. Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect PIK3CA mutations in human genomic DNA. The mutant PIK3CA gene is amplified by the specific primers, and detected by the novel probes. Protocol The mutation assay for each sample and control assay must be analyzed within the same PCR run to avoid run-to-run variations in threshold settings. It is recommended that the PIK3CA Mixed Standard (STD)should be analyzed during each PCR run, along with no-template controls (NTC). 1. Thaw PIK3CA Reaction Mixes and Mixed Standard. Then centrifuge PIK3CA Taq DNA polymerase, Reaction Mixes and Mixed Standard prior to use. 2. Accordingtotheratioof0.2μL PIK3CA Taq DNA Polymerase to 20 μl E542K or E545K Reaction Mix per sample, and 0.16 μl PIK3CA Taq DNA Polymerase to 20 μl other Reaction Mix (or External Control Reaction Mix) per sample, transfer the appropriate amount of PIK3CA Taq DNA Polymerase and Reaction Mixes (or External Control Reaction Mix) into a sterile tube. Mix each solution for approximately 15 seconds and then centrifuge for 15 seconds. 3. Transfer 20 μl of the above master mix into PCR reaction wells. 4. Add 5 μl sample DNA (see following for sample DNA concentrations), 5 μl PIK3CA Mixed Standard or 5 μl ddh2o (no-template control) to the appropriate PCR reaction wells (Table 3). 5. According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens. a) Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood. i. For non-paraffin embedded samples, the recommended DNA amount in each test tube is 2 ~ 5 ng. a) For paraffin embedded samples, we recommend use of 10 ~ 15 ng template DNA in each PCR tube based on different storage times. i. Use 10 ng of template DNA for samples with less than 3 years storage time. ii. Use 15 ng of template DNA for samples with more than 3 years storage time. Notes: We recommend use of TE (ph = 8.0) for extracted DNA dilution. Since Taq DNA polymerase is viscous, please pay attention to the centrifugation and pipetting process. Minimize the contact interface between the pipette tip and Taq DNA polymerase to avoid adding excess enzyme. 6. Seal the PCR tubes. 7. Spin the PCR tubes gently to collect the reagents at the bottom of wells. Note: This spin step is essential for proper mixing of the reagents. 8. Place the PCR tubes into the real-time PCR instrument. 9. Carry out real-time PCR using the cycling conditions described in Table 4. 3/5
4 Table 3 Plate Layout (example for 14 tests) For:ADx-PI Sample1 Sample1 Sample1 Sample1 Sample1 Sample1 Sample9 Sample9 Sample9 Sample9 Sample9 Sample9 2 Sample2 Sample2 Sample2 Sample2 Sample2 Sample2 Sample10 Sample10 Sample10 Sample10 Sample10 Sample10 3 Sample3 Sample3 Sample3 Sample3 Sample3 Sample3 Sample11 Sample11 Sample11 Sample11 Sample11 Sample11 4 Sample4 Sample4 Sample4 Sample4 Sample4 Sample4 Sample12 Sample12 Sample12 Sample12 Sample12 Sample12 5 Sample5 Sample5 Sample5 Sample5 Sample5 Sample5 Sample13 Sample13 Sample13 Sample13 Sample13 Sample13 6 Sample6 Sample6 Sample6 Sample6 Sample6 Sample6 Sample14 Sample14 Sample14 Sample14 Sample14 Sample14 7 Sample7 Sample7 Sample7 Sample7 Sample7 Sample7 STD STD STD STD STD STD 8 Sample8 Sample8 Sample8 Sample8 Sample8 Sample8 NTC NTC NTC NTC NTC NTC Table 4 Cycling Parameters Temperature Time Cycles Stage min 1 Stage s s s Stage s s Data collection of FAM and s Note:The probe settings on ABI machine: Reporter Dye:VIC;Quencher Dye:TAMRA;Passive Reference:NONE Sample Data Analysis 1. The FAM signals of the mutation detection system indicate the mutation status of the sample. The signals indicate the internal control status. The internal control amplifies and detects a region of genomic DNA adjacent to the PIK3CA gene. 2. Check the FAM signals from the external control assay: i. The Ct value should be between 15 ~ 22 for paraffin embedded specimens; and between 13~19 for non-paraffin embedded specimens. ii. If the requirements of i) are satisfied, further analysis should be carried out. However, if Ct value is below the indicated range, the DNA is overloaded. The procedure should be repeated with reduced DNA. iii. If the external control assay has failed, the DNA template contains PCR inhibitors, indicting that the DNA needs to be re-extracted. 3. The signals of the internal control are also used as controls. If the signals assay failed but the FAM test worked well, please continue with the analysis. If both the and FAM signals failed, the data should be discarded to repeat the experiment. 4. Instrument set-up: Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose reaction holes for positive control, no-template reference and samples simultaneously. Then, users may adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 5. The PIK3CA mixed standard FAM Ct value should be less than 20, but variation may occur due to different threshold settings on different instruments. Analysis of mutation assay results. See Table Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are divided into strong positive, weak positive or negative 4/5
5 2. Strong Positive: If the sample FAM Ct value is less than the Ct value shown in the Strong Positive row in Table5, the sample is classified as strong positive. 3. Weak Positive: If the sample FAM Ct value is in the range shown in the Weak Positive row in Table 5, the sample is provisionally classified as weak positive. a. If the FAM Ct value is in the Weak Positive range, the Ct of the reaction tube is calculated to confirm the result. b. If the Ct value is less than the corresponding Cut-off value of Ct, the sample is confirmed as weak positive. c. If the Ct value is greater than or equal to the Cut-off Ct value, the sample is classified as negative or below the detection limit of the kit. 4. The calculation of Ct: Formula 1. Ct = mutant FAM Ct value external control FAM Ct value. a. Where: i. The mutant FAM Ct value indicates the Ct value of the mutant FAM signals from a sample. ii. The external control FAM Ct value indicates the Ct value of the FAM signal in external control tube. 5. Negative: If the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative row in Table5, the sample is classified as negative or below the detection limit of the kit. Table 5 Results Determination Name of Mutation H1047R H1047L E542K E545K E545D Strong Mutant Ct Positive Value Ct<25 Ct<26 Ct<25 Ct<26 Ct<26 Mutant Ct Weak Value 25 Ct <28 26 Ct <29 25 Ct <29 26 Ct <29 26 Ct <29 Positive Ct Cut-off value Negative Mutant Ct Value Ct 28 Ct 29 Ct 29 Ct 29 Ct 29 Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. Do not exchange and mix up the kit contents with different batches. 3. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx. 4. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 5. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. 6. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 7. All the chemicals are potential hazard, only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References 1. Bunney TD & Katan M, Nature Rev. Cancer. 10: Yap TA, et al Curr Opin Pharmacol. 8: Sartore-Bianchi A, et al Cancer Res. 69: Serra V, et al Cancer Res. 68: /5
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