QGENEFV. Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat.

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1 Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat.# G0102 Institute of Applied Biotechnologies a.s. User manual Version 04/2011

2 OBSAH QGENEFV 1. S T OR A G E CONTENTS INFORMATION ABOUT THE MUTATION IN FACTOR V GENE KIT DESCRIPTION KIT PRINCIPLE ADDITIONALLY REQUIRED MATERIALS AND DEVICES GENERAL PRECAUTIONS PROTOCOL DNA ISOLATION PCR PREPARATION SETTINGS OF ROTORGENE ANALYZER DATA ANALYSIS TROUBLESHOOTING SPECIFICATION ANALYTICAL SENSITIVITY ANALYTICAL SPECIFICITY INFORMATION FOR WASTE DISPOSAL

3 1. S t or a g e All kit components must be stored at -20 C or lower. Under this conditions kit is stable until the expiration date stated on the label. 2. Contents MasterMix transparent 0.5 ml tube with purple cap (8 x 12 reactions) PCR H 2 O transparent 0.5 ml tube with blue cap (50 µl) Control Sample: Heterozygous sample transparent purple 0.2 ml tubes (8 x 10 µl) 3. Information about the Mutation in Factor V gene Venous thrombosis is clinically serious disease with high incidence of % in developed countries. The genesis is influenced by many factors both clinical (obesity, surgery) and genetic (mutations in genes encoding for Factor C, protein S, anti-thrombin, Factor V and prothrombin). The mutation in Factor V gene (Leiden mutation) is the most significant mutation of the genetic factors associated with the venous thrombosis. and it leads to the failure of anticoagulant system. The substitution of guanine for adenine on the position 1691 (G1691A) of the gene encoding Factor V results in the substitution of glutamine for arginine on the position 506 of the Factor V protein. It leads to the resistance to the anticoagulant effect of activated protein C that is necessary to the degradation of Factor V to Factor VII. Heterozygous form of Factor V (Leiden) increases the risk of thrombosis 3 8 times, homozygous mutant form up to 80 times. The congenital risk of thrombophilic factors including the mutation in Factor V gene are responsible for some serious states in the pregnancy and those failures compared to healthy pregnant population. By the recent studies the mutation in Factor V 3

4 gene has the significant influence of the repeated spontaneous abortions in the first trimester of the pregnancy. Detection kit for mutation G1691A in Factor V gene is based on quantitative real-time PCR analysis that saves time and makes possible the analysis of huge amounts of samples. The kit is suitable for routine diagnostics. The results are not influenced by oral coagulation therapy and are prognostic parameters for patients with an inclination to thrombosis. 4. Kit Description Kit is designed to determine mutation G1691A in Factor V gene using Rotor- Gene Analyzer 3000/6000/Q. Kit contents all components required for specific amplification and direct detection of gene fragment for factor V carrying either mutated or healthy sequence. Each kit contains control heterozygous sample for the verification of the PCR process. 5. Kit Principle Kit for detection of mutation G1691A in Factor V gene is based on the amplification of the specific region by the PCR method. The real-time technology is possible to view the increase in the amount of DNA as it is amplified. Two independent fluorescent signals are detected in each tube. 4

5 6. Additionally Required Materials and Devices Disposable powder-free gloves Adjustable pipettes Sterile pipette tips with filters Vortex mixer Benchtop centrifuge Rotor-Gene Analyzer (Corbett Research) Cooling blocks for 0.1 ml and 1.5 ml tubes 0.1 ml strips for 72-well rotor or 0.2 ml tubes (Corbett Research) 7. General Precautions The user should always pay attention to the following: 1. Use sterile pipette tips with filters 2. Store and extract positive materials separately from all other components 3. Add the positive material to the reaction mix in a spatially separated facility to avoid contamination 4. Work quickly, keep all reagents in a cooling block 8. Protocol 8.1. DNA isolation Currently there are different DNA isolation kits and it is always necessary to proceed according to manufacturers protocol. It is recommended to use DNA samples containing ng/µl of DNA and its purity has to be in the range of when determined spectrophotometrically by the A260/A280 ratio. It is advised to use for extraction NucleoSpin Blood Kit (Macherey-Nagel). 5

6 8.2. PCR Preparation Before the preparation of PCR reactions it is necessary to perform the following steps: 1. Make sure that the cooling block is pre-cooled to +4 C. 2. Prepare the desired number of PCR tubes in the pre-cooled block. 3. Thaw tubes with MasterMix and PCR H 2 O that will be used for the reactions at +4 C. One tube of MasterMix contains 12 reactions. Is it denied to freeze the tube with MasterMix again and use it later. 4. Let MasterMix and PCR H 2 O thaw in a fridge, mix them up by quick vortexing and centrifuge briefly. 5. Prepare corresponding number of PCR tubes a place them to the cooling block (+4 C). PCR setup: MasterMix 13 µl Template 2 µl Always use the negative control (MasterMix and PCR H 2 O) and the heterozygous control samples. Repeated thaw/freezing of applied control sample is not recommended. Close the PCR tubes properly and place them into the rotor of Rotor-Gene Analyzer. Temperature profile 95 C 24 min 95 C 15 s 62 C 30 s 40 x, acquiring in Green, Yellow Gain optimalization: Green 5-10Fl, Yellow 1-5Fl 6

7 8.3. Settings of RotorGene Analyzer CD containing the template/setting file trombofilni_mutace_3000.ret is part of the first delivery of the kit. All information necessary to automatic adjustment of the RotorGene analyzer is saved in that file. Turn on the RotorGene Analyzer. Place the CD into the CD-ROM drive of your PC and start the software for the RotorGene Analyzer by double clicking on the icon: A dialogue window will be opened after start the application or clicking on icon New. Select the tab Advanced in this dialog window and the item Open A Template In Another Folder and search the required file on CD to open. 7

8 Select the rotor type (72-well rotor) and tick the Locking Ring Attached. Write your name to the field Operator and eventually your own notes to the experiment to the field Notes. Click the button Next to reach the window with the button Start Run and click this button. 8

9 Choose the directory for the saving of the experiment and set the name of the experiment file. The experiment will be started after clicking the button Save. The last window of experiment settings serves to identify of each samples. 9

10 9. Data analysis Start the analysis by the button Analysis (1) after the finishing of PCR reaction. Open the Allelic Disc tab (2). If this item isn t available, you find it in Other tab. Now select channels Green (Cycling A. Green) and Yellow (Cycling A. Yellow) and press Show (3). In the software for Rotor Gene 3000 are these channels called FAM (Cycling A.FAM/Sybr) and JOE (Cycling A.JOE)

11 The window containing graph displaying values of normalized fluorescence as a function of PCR cycles is opened. Check that functions Dynamic Tube (1) and Slope Correct (2) are activated. 1 2 Set value for Threshold in range of 0,05-0,2. If necessary, verify the results in the raw data. 11

12 Example of evaluation of results: Channel Green Channel Yellow Evaluation Healthy sample Mutated sample Heterozygous sample 12

13 Evaluation: Genotypes for each samples will be obtained after setting a value Treshold in the window Allelic Discrimination Results You can print obtained results using the icon Reports. 13

14 10. Troubleshooting No signal with any/some samples. Incorrect preparation of the reaction. Check all steps of preparation of PCR reaction and repeat the PCR, if necessary. The storage conditions for kit components did not comply with the instructions given in Storage (Chapter 1). Please check the storage conditions, the expiration date. Use the new kit, if necessary. Always check that the kit wasn t delivered thawed. Make sure that you used recommended amount of the DNA. Measure the concentration of DNA samples and repeat the PCR with the right amount of DNA. 11. Specification 11.1 Analytical Sensitivity The minimal amount of DNA is 40 ng per reaction (the DNA concentration 20 ng/µl) Analytical Specificity Analytical specificity is given by the selection of primers and probes. The homologies of these probes were checked with the reference sequence in the database National Center for Biotechnology Information. Results of statistically valuable group of samples were confirmed by sequencing. 12. Information for waste disposal The waste disposal of all used material has to be done according to the internal instructions of user s institute. If you have any problems, please contact our technical support at. 14

15 Institute of Applied Biotechnologies a.s. Praha 10 Produced by Institute of Applied Biotechnologies a.s. Tymiánová 619/14 Praha 10 IČO: Last revision date

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