AmoyDx TM JAK2 V617F Mutation Detection Kit Detection of V617F mutation in the JAK2 oncogene Instructions For Use Instructions Version: B1.0 Date of Revision: July 2012 Store at -20±2 o C -1/5-
Background The JAK family of non-receptor tyrosine kinases, includes JAK1, JAK2, JAK3, andtyk2. Growth factors and cytokines can regulate gene transcription through JAK-dependent activation of signal transducer and activator of transcription (STAT). The JAK-STAT signal transduction pathway regulates cell proliferation, differentiation, apoptosis and immune regulation. Activation of JAK2 by mutation of the amino acid at position 617 (V617F ) is associated with myeloproliferative disorders, including polycythemia vera (mutations found in 90% of cases), essential thrombocythemia (50%) and idiopathic myelofibrosis (50%). JAK2 mutation is the main diagnostic indicator of MPD. The ADx JAK2 V617F Mutation Detection Kit is a highly selective and sensitive assay for detecting the V617F mutation in the JAK2 oncogene. AmoyDx s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA, while ensuring that false negatives are minimized. The procedure is easily adapted for use in high-throughput sample processing. Intended Use AmoyDx JAK2 V617F Mutation Detection Kit is intended for research use only. Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 1), and additional mixed standard DNA (genomic DNA plus V617F mutated plasmid DNA) for positive control reactions. Table1 Kit Contents Tube Reagents Supplied 24 reactions Volume 1 V617F Reaction Mix 800 μl 2 JAK2 Taq DNA Polymerase 15 μl 3 JAK2 Mixed Standard 150 μl Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instrument is Rotor-gene 6000. 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling DNA template. 4. Sterile, nuclease-free H2O. Shipping and Storage The kit requires cold-chain-transportation. The shelf-life of the kit is eight months when the kit is stored immediately upon receipt at -20±2 o C in a constant-temperature freezer and protected from light. Specimen Material Human genomic DNA must be extracted from non-heparin blood prior to use and stored at -20±2 o C. Good DNA quality is essential and we recommend use of Qiagen DNA extraction kits (DNeasy Blood & Tissue kit, cat. No. 69504 or 69506). 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 the JAK2 V617F mutation in -2/5-
human genomic DNA. The mutant JAK2 V617F gene DNA is amplified by the specific primers, and detected by the novel probes. Protocol Notes: 1. Each reaction tube includes a mutation detection system and internal control system. The mutation detection system is used to detect the mutation status of the JAK2 gene. The internal control system is designed for detecting the presence of inhibitors, which may lead to false negative results. 2. The threshold at which the signal is detected above background fluorescence is called the Cycle threshold (Ct). The Ct values used to determine if a sample is positive or negative are based on extensive validation. If the Ct value falls within the appointed range (see below), the sample is classed as mutation positive. If the Ct value is outside the appointed range, the sample is classed as negative or below the detection limit of the kit. 3. The JAK2 mixed standard contains a recombinant JAK2 gene with the V617F mutation, and normal human genomic DNA. 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 JAK2 mixed standard (STD) should be analyzed during each PCR run, along with no-template controls (NTC). 1. Thaw the Reaction Mixture. 2. Centrifuge JAK2 Taq DNA polymerase and Reaction Mixture prior to use. 3. Add 0.25 μl Taq DNA polymerase to 25 μl reaction mixture per sample. 4. Mix the solution by gently pipeting it up and down about 20 times. a) The Taq enzyme should avoid vortexing. 5. Centrifuge briefly. 6. Transfer 25 μl solution into appropriate PCR tubes. 7. Add 5 μl sample DNA (1~2 ng/µl), 5 μl JAK2 mixed standard or 5 μl ddh2o(no-template control,ntc) to the appropriate PCR tubes. The layout for 22 samples, a positive control and a no-template control. 8. Seal the PCR tubes. 9. Place the PCR tubes into the real-time PCR instrument. 10. Carry out real-time PCR using the cycling conditions described in Table 2. Note 1: Prior to the operation, please set up the PCR program by the following steps: 1select Gain Optimisation, then Auto Gain Optimisation Setup window will open; 2Click on Optimise Acquiring and Perform Calibration Before 1st Acquisition, Tube 1 is used as control. 3Adjust the temperature to 56, and click Start to continue. Please see the following Figure 1, Figure 2 and Figure 3 for more details. Note 2: Make sure the total volume of solution in each well is 30μl (25μlreagentplus5μl DNA) Table 2 Cycling Parameters Temperature Time Cycles Stage 1 95 5min 1 Stage 2 95 25s 60 20s 15 72 20s Stage 3 93 25s 56 35s Data collection of FAM and HEX 26 72 20s Figure 1-3/5-
Figure 2 Figure 3 Sample Data Analysis 1. The FAM signal indicates the mutation status of sample and the HEX signal indicates the internal control status. The HEX control amplifies and detects a region of genomic DNA adjacent to the JAK2 gene. 2. Make sure that each well gives a HEX signal. If the HEX signal gives a positive result, then continue with the analysis. If the Ct value <13, it indicates that the DNA was overloaded, and the amount of DNA should be reduced. If the HEX signal assay failed, it shows that the DNA template contains PCR inhibitors. In this case, the DNA should be re-extracted and the whole experiment should be carried out again. 3. Ensure the calibration fluorescence is unselected,select the sample and control positions as a group. Then adjust the Threshold for FAM amplification curves to obtain the Ct values of the samples and controls. 4. The mixed standard FAM Ct value should be less than 21, but variation may occur due to different threshold settings on different instruments. 5. Analysis of mutation assay results. a) 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. b) Negative: If the sample FAM Ct value is greater than or equal to 26 (the critical negative value), the sample is classified as negative or below the limits of the kit. c) IftheFAMCtvalueislessthan26(thecriticalnegative value), please analyze the results by the following approaches: i) Strong Positive: If the sample FAM Ct value is less than 23 (critical positive value), the sample is classified as strong positive. ii) If the sample FAM Ct value is greater than or equal to 23 (critical positive value), the Ct of the reaction tube is calculated to confirm the result. If the Ct value is less than 5, the sample is confirmed as weak positive.ifthe Ct value is greater than 5, the sample is classified as negative or below the limits of the kit. d) The calculation of Ct: Ct = mutant FAM Ct value internal control HEX Ct value. The mutant Ct value indicates the Ct value of the sample mutant FAM signal; the internal control Ct value indicates the Ct value of internal control HEX signal of the sample. 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. -4/5-
6. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 7. 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 the 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. Goldin LR, et al. Genome Med. 2009. 1:55. 2. Tefferi A, Leuk Lymphoma. 2008. 49:388-97. 3. Kilpivaara O, Nat Genet. 2009. 41: 455-9. -5/5-