MBS598266 - MyBioSource
Contents
1. 15sec 60 C for 1min Fluorescence measured at 60 C 40cycles 5 r you use ABI Prism system please choose none as passive reference 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 Molecular Grade Water UNDET 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 peta Gis UNDET Below the detection limit or negative Positive and the software displays the quantitative value 38 40 Retest if it is still 38 40 report as 1 For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES2. 2 Positive Control Attention It is necessary to dilute the positive control supplied in the kit to 10 copies ml by 10 times with molecular grade water before detection and close the tube immediately then vortex for 10 seconds 9 3 Quantitation The kit can be used for quantitative or qualitative real time RT PCR 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 1x10 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 Dilution of Standards 4yl Aul Aul fones 1X107 1X10f 1X10 1K 1D copiessmt 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 contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 4 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 14 1 pl Super Mix Enzyme Mix Spl 15yl Extraction RNA Master Mix Reaction Plate Tube PCR Instrument 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the
3. number of samples which includes the number of controls standards 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 2 Pipet 15u Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 5al 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 10 min 1 cycle 95 C for 15 min 1 cycle 95 C for 5 sec 60 C for 30sec 40 cycles Fluorescence is measured at 60 C channel FAM should be chosen 10 Baseline setting just above the maximum level of molecular grade water 11 Calabration 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 The crossing point value of molecular grade water and positive control in FAM channel shows blank and lt 35 respectively Correlation coefficient of standard curve should be lt 0 98 otherwise the result is invalid 13 Data Analysis and Interpretation The following results are possible 1 The crossing point value in channel FAM shows lt 38 The result is posi
4. of serum AFP provides a useful marker for diagnosis and prognosis of these tumors However the serum AFP level does not always correspond to the clinical stage of HCC Recent molecular biological techniques have provided a method for detecting malignant cells in the peripheral blood by amplification of messenger RNA mRNA of various genes specific to a particular cell type from peripheral blood mononuclear cells AFP mRNA has been demonstrated to be one of the candidate molecules for detecting HCC cells in the blood AFP mRNA in the peripheral blood of patients with hepatocellular carcinoma HCC may indicate hematogenous spread of HCC AFP mRNA real time RT PCR kit contains a specific ready to use system for the detection of the AFP mRNA using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains three Super Mixes for the specific amplification of M BCR m BCR and u BCR The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the AFP mRNA 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 BCR ABL fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 An external positive control 1 lt 10 copies ml
5. EU C Revision No ZJ0006 Issue Date Jul 1 2015 AFP mRNA Expression in Peripheral Blood Real Time RT PCR Kit MBS598266 Instrument I II For Use with LightCycler 1 0 LightCycler2 0 Real Time PCR Systems For Research Use Only In USA amp China User Manual 1 Intended Use AFP mRNA real time RT PCR Kit is used for the detection of AFP mRNA in mononuclear cell 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 Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description a Fetoprotein AFP a serum protein produced in large amounts during fetal life rapidly reduces from late fetal life and is essentially scarce in normal adults The synthesis of AFP is often associated with the development of HCC and yolk sac tumors The detection
6. R 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 a Fetoprotein AFP a serum protein produced in large amounts during fetal life rapidly reduces from late fetal life and is essentially scarce in normal adults The synthesis of AFP is often associated with the development of HCC and yolk sac tumors The detection of serum AFP provides a useful marker for diagnosis and prognosis of these tumors However the serum AFP level does not always correspond to the clinical stage of HCC Recent molecular biological techniques have provided a method for detecting malignant cells in the peripheral blood by amplification of messenger RNA mRNA of various genes specific to a particular cell type from peripheral blood mononuclear cells AFP mRNA has been demonstrated to be one of the candidate molecules for detecting HCC cells in the blood AFP mRNA in the peripheral blood of patients with hepatocellular carcinoma HCC may indicate hematogenous spread of HCC AFP mRNA real time RT PCR kit contains a specific ready to use system for the detection of the AFP mRNA using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains three Super Mixes f
7. X107 1X10f 1X10 1X10fcopiesmi 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 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 194l 1 yl Super Mix Enzyme Mix 5ul 204 Extraction RNA Master Mix Reaction Plate Tube 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 standards 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 2 Pipet 20ul AFP 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 10min 95 C for 15min Selection of fluorescence channels Target Nucleic Acid 95 C for
8. em e Real time PCR reaction tubes plates e Pipets 0 5ul 1000u1 e Sterile microtubes 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 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 Aul Au I 4 u 1
9. g 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 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 Nucleic Acid Isolation Kit Cat Number Manufacturer RNA Isolation Kit ME O010 ME 0012 ZJ Biotech _ QlAamp Viral RNA Mini extraction Kit 50 52904 QIAGEN 9
10. or the specific amplification of M BCR m BCR and u BCR The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the AFP mRNA 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 BCR ABL fragment is performed in fluorimeter channel FAM 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 3 Quantitation 4 Kit Contents AFP Super Mix 1 vial 480u1 1 vial 28ul 1 vial 400ul 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1 X 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 a
11. supplied allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents AFP Super Mix 1 vial 380u1 1 vial 28 ul 1 vial 400u1 1 vial 30u1 RT PCR Enzyme Mix Molecular Grade Water AFP Positive Control 1 lt 10 copies ml 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 Warnings and Precaution 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 accordin
12. tive The sample contains AFP mRNA 2 The crossing point value in channel FAM shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative 3 In channel FAM no signal is detected The sample does not contain any AFP mRNA It can be considered negative For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES IVD Revision No ZJ0008 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China AFP mRNA Expression in Peripheral Blood Real Time RT PCR Kit User Manual 20 C MBS598266 Instrument II IV Z 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 Ed Bod 1 Intended Use AFP mRNA real time RT PCR Kit is used for the detection of AFP mRNA in mononuclear cell 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 PC
13. voided 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 Ta 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 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 RT PCR Enzyme Mix Molecular Grade Water AFP Positive Control 1 lt 10 copies ml e Real time PCR syst
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