MBS598288 - MyBioSource
Contents
1. Do not use the lymphocytes separation media to obtain leukocytes The leukocytes precipitate obtained can be directly used for RNA extraction and it can also be dissolved in RNA extraction reagents such as Trizol RLT buffer for long time storage at 80 C It s strongly recommended not to store the leukocytes precipitate without any RNA extraction reagent 9 1 2 RNA extraction from leukocytes RNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kits based on the yield For the RNA extraction please comply with the manufacturer s instructions The recommended extraction kit is as follows e g RNA extraction with Trizol 1 Add 1 ml Trizol into the leukocytes and pipet up and down several times to make cells fully dissolved Increase the volume of Trizol proportionately if leukocytes are more than 5 X 10 cells 2 Add 0 2 ml chloroform and shake the tube by vortex for 15 sec at least 3 incubate the tube at room temperature for 2 3 min 4 Centrifuge the sample at 13 000 rpm for 15 min at 4 C The following procedures should be operated on ice box 5 Transfer the aqueous phase above approximately 0 4 0 6 ml into a new 1 5 ml centrifuge tube avoiding disturbing any of the white interphase 6 Add pre cooled isopropanol into the aqueous phase and mix by pipetting up and down for 10 times 7 Incubate for 1 hour at 20 C 8 Centrifuge at 13 000 rpm for 15 min a2. 43 45 report as 1 For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Selection of fluorescence channels Target Nucleic Acid EU C Revision No ZJ0003 Issue Date Jul 1 2015 For Research Use Only In USA amp China Leukemia WT 1 Gene Real Time RT PCR Kit User Manual 20 C MBS598288 Instrument HI IV 25 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument Ea tod 1 Intended Use Leukemia WT 1 gene real time RT PCR Kit is used for the detection of Leukemia WT 1 gene in leukocyte by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumul
3. J IVD Revision No ZJ0002 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Leukemia WT 1 Gene Real Time RT PCR Kit 20 C 29 User Manual MBS598288 Instrument I IT For use with LightCycler1 0 2 0 Instrument e frer 1 Intended Use Leukemia WT 1 gene real time RT PCR Kit is used for the detection of Leukemia WT 1 gene in leukocyte by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description Wilms tumor gene WT1 encodes a transcription factor and plays an important role in cell growth and differentiation The WT1 gene is highly expressed in leukemia and various types of solid tumors whereas WT1 is a tumor marker convenient for the detection of minimal residual di
4. R reaction plate tubes Separately add 5ul RNA sample positive and negative controls to different plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 45 C for 20min Icycle 95 C for 5min Icycle 95 C for S5sec 58 C for 20 sec 72 C for 35 sec Fluorescence measured at 58 C 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the Noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control AScycles 11 Calibration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Negative control positive control QS and must be performed correctly otherwise the sample results is invalid Crossing point value Molecular Grade Water Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible _ Crossing point value Result Analysis Below the detection limit or negative 2 lt 43 Positive the sample contains WT 1 gene transcripts and the software displays the quantitative value 43 45 Re test If it is still
5. ade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR Take positive control 110 copies ml as the starting high standard in the first tube Respectively pipette 36ul of Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul Au l 4u l 1X107 1X10 1X10 1X 104 copiesim To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 1 lt 10 copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 14 ul 1 yl Super Mix Enzyme Mix Spl 154l Extraction RNA Master Mix Reaction Plate Tube l PCR Instrument 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge Mix completely then spin down briefly in a centrifuge 2 Pipet 15ul Master Mix with micropipets of sterile filter tips to each of the real time PC
6. ating product without having to re open the reaction tube after the amplification 3 Product Description Wilms tumor gene WT1 encodes a transcription factor and plays an important role in cell growth and differentiation The WT1 gene is highly expressed in leukemia and various types of solid tumors whereas WT1 is a tumor marker convenient for the detection of minimal residual disease of leukemia The WT1 gene was originally defined as a tumor suppressor gene but we proposed that it was on the contrary an oncogene Leukemia WT 1 gene real time RT PCR kit contains a specific ready to use system for the detection of the Leukemia WT 1 gene using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Leukemia WT 1 gene is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified WT 1 fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 An external positive control 1 lt 10 copies ml supplied allows the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents Type of reagent WT 1 Super Mix 1 vial 480
7. eated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kit based on the yield For the RNA extraction please comply with the manufacturer s instructions The recommended extraction kit is as follows 9 2 Quantitation The kit can be used for quantitative or qualitative real time RT PCR A positive control defined as 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Gr
8. ference and quencher 10 Threshold setting just above the maximum level of molecular grade water 11 Calibration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Negative control positive control and QS curve must be performed correctly otherwise the sample results is invalid QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value Result Analysis UNDET Below the detection limit or negative 2 lt 43 Positive the sample contains WT 1 gene transcripts and the software displays the quantitative value 43 45 Re test If it is still 43 45 report as 1 For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
9. s should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 1 Leukocytes separation e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000p1 e Sterile microtubes You can use commercial erythrocytes lysis buffer to remove the erythrocytes from blood samples Please refer to the specific instructions for erythrocytes lysis buffer Attention
10. sease of leukemia The WTI gene was originally defined as a tumor suppressor gene but we proposed that it was on the contrary an oncogene Leukemia WT 1 gene real time RT PCR kit contains a specific ready to use system for the detection of the Leukemia WT 1 gene using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Leukemia WT 1 gene is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified WT 1 fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 An external positive control 1x10 copies ml supplied allows the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents Type of reagent WT 1 Super Mix 1 vial 3801 RT PCR Enzyme Mix 1 vial 28 ul Molecular Grade Water 1 vial 400ul WT 1 Positive Control 1 10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the RNA extraction kits recommended the analysis sensi
11. sfer B The positive control 1x10 copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 19 1 ul 1 The volumes of Super Mix and Enzyme Mix per reaction Super Mix Enzyme Mix multiply with the number of biii a pena i samples which includes the number of controls and sample Spl 20pl prepared Molecular Grade Water Extraction RNA Master Mix is used as the negative control For reasons of unprecise i ee pipetting always add an extra Reaction virtual sample Mix completely late Tube then spin down briefly in a Plate Tu centrifuge Mix completely then spin down briefly in a centrifuge PCR Instrument 2 Pipet 20n1 Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 5ul RNA sample positive and negative controls to different plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 45 C for 20min Selection of fluorescence channels et Nucleic Acid 95 C for Smin 95 C for 15sec 58 C for 30 sec 72 C for 45 sec AScycles Fluorescence measured at 58 C 5 A If you use ABI Prism system please choose none as passive re
12. t 4 C and carefully remove the supernatant from the tube 9 Add pre cooled 75 ethanol and gently pipet RNA pellet 10 Centrifuge the sample at 13 000 rpm for 15 min at 4 C and carefully remove the supernatant from the tube avoiding disturbing the RNA pellet 11 Add 40 ul DEPC H 0 to the RNA pellet after Air drying for 5 10 min then shake gently 12 Centrifuge instantaneously and incubate for 10 min at room temperature to make RNA fully dissolved Extracted RNA can be used for following PCR reactions immediately or stored at 80 C for long time 9 2 Quantitation The kit can be used for quantitative or qualitative real time RT PCR A positive control defined as 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR Take positive control 110 copies ml as the starting high standard in the first tube Respectively pipette 36ul of Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards To generate a standard curve on the Au 4 ul Aul real time system all four dilution WU i N standards should be used and defined as standard with specification of the corresponding N concentrations y y Attention y y A Mix thoroughly before next 1X107 1X10 1X10 1X10 copiesm tran
13. tivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e RNA extraction kit e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 pl e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and freezer e Tube racks 7 A Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid rep
14. u1 RT PCR Enzyme Mix 1 vial 28 ul Molecular Grade Water 1 vial 400ul WT 1 Positive Control 1 10 copies ml 1 vial 30u1 Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the RNA extraction kits recommended the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g A 7 A Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical sample
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