Time is short Run a HemaVision®-28Q
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1. DNA DIAGNOSTIC IIS la delt p32 STIL TAL1 The list is not 1 11 p32 q23 MLL EPS15 1 11 921 923 MLL MLLT11 1 19 923 13 TCF3 PBX1 3 5 q25 q34 NPM1 MLF1 3 21 q26 q22 RUNX1 MECOM 4 11 421 423 MLL AFF1 5 12 q33 p13 ETV6 PDGFRB 5 17 q35 q21 NPM1 RARA 6 9 p23 q34 DEK NUP214 6 11 927 923 MLL MLLTA AME 8 21 q22 q22 RUNX1 RUNX1T1 t 9 9 q34 q34 SET NUP214 HemaVision 28Q t 9 11 22 423 MLL MLLT3 28 Translocations 9 12 934 13 ETV6 ABL1 145 9 22 q34 q11 BCR ABL1 10 11 12 423 MLL MLLT10 In just 4 hours 11 17 q23 q21 MLL MLLT6 11 17 423 421 ZBTB16 RARA 632 iD 11 19 923 13 1 MLL ELL 11 19 423 13 3 MLL MLLT1 12 21 p13 q22 ETV6 RUNX1 12 22 p13 q11 6 1 15 17 924 921 PML RARA inv 16 13 422 CBFB MYH11 16 21 11 422 FUS ERG 17 19 922 13 TCF3 HLF t X 11 q13 q23 MLL FOXO4 Cat No HV01 28Q USER MANUAL Revision date 2015 09 30 g DNA DIAGNOSTIC USER MANUAL HEMAVISION 28Q Cat 01 280 MULTIPLEX RT QPCR TEST FOR SCREENING OF 28 LEUKEMIA ASSOCIATED TRANSLOCATIONS 12 TESTS PER KIT C IVD FOR PROFESSIONAL USE ONLY MANUFACTURER DNA Diagnostic A S Voldbjergvej 16 8240 Risskov Denmark Homepage www dna diagnostic com Email info dna diagnostic com Phone 0045 87323050 TRADEMAR2. cs t 11 19 q23 p13 3 MLL ex8 9 MLLT1 ex2 t 4 11 q21 q23 MLL ex8 9 AFF1 ex9 t 17 19 q22 p13 TCF3 ex14 HLF ex4 del 1 p32 STIL ex1 TAL1 ex1 t 9 22 q34 q11 BCR ex1 ABL1 ex3 t 9 9 q34 q34 SET ex9 NUP214 ex19 t 11 19 q23 p13 3 MLL ex7 MLLT1 ex9 t 9 22 q34 q11 BCR ex12 ABL1 ex3 ROX t 9 22 q34 q11 BCR ex19 ABL1 ex3 t 11 17 q23 q21 ZBTB16 ex4 RARA ex3 ROX CYS 16 Reference gene ABL1 ex2 ABL1 ex3 CY5 t 9 12 q34 p13 ETV6 ex2 5 ABL1 ex3 FAM CY5 t 5 12 q33 p13 ETV6 ex2 5 PDGFRB ex12 ROX 5 t 10 11 p12 q23 MLL ex8 9 MLLT10 ex20 FAM CY5 t 1 11 q21 q23 MLL ex8 9 MLLT11 ex2 ROX CY5 t X 11 q13 q23 MLL ex7 FOXO4 ex2 cys t 11 17 q23 q21 MLL ex7 MLLT6 ex12 ROX CY5 FAM 5 o t 3 21 q26 q22 RUNX1 MECOM RUNX1 ex6 MECOM ex2 t 10 11 p12 q23 MLL MLLT10 MLL ex7 MLLT10 ex9 CYS t 5 17 q35 q21 NPM1 RARA NPM1 ex4 RARA ex3 FAN CY5 21 t 3 5 q25 1 q35 NPM1 MLF1 1 ex4 MLF1 ex4 t 10 11 p12 q23 MLL MLLT10 MLL ex7 MLLT10 ex13 CY5 t 3 21 q26 q22 RUNX1 MECOM RUNX1 ex9 t 10 11 p12 q23 MLL MLLT10 MLL ex8 MLLT10 ex12 gt gt gt gt gt gt gt N x 1 LAD HemaVision 28Q When more than one of the tubes 1 6 8 15 17 23 are positive for FAM or ROX interpretate as follows Positive tubes Interpretate as tube 1 ROX 1 ROX Inv 16 CBFB MYH11 2 FAM 1 FAM 3 FAM 3 FAM t 15 17 PML RARA bcr1 L form 4 FAM 1 F
3. 585 nm Em 605 nm CY5 Abs 635 nm Em 665 nm e Pipettes and sterile RNase free filtered tips Gloves HemaVision 28Q 5 PRECAUTIONS e The quality and concentration of the RNA sample greatly affects the results of this test To minimize the risk of degradation by ribonucleases we strongly recommend purification of total RNA immediately after blood bone marrow extraction Do not freeze Ficoll purified cells without adding a denaturing solution e g containing guanidinium isothiocyanate GTC immediately after isolation and before freezing Always store cell samples and aqueous RNA solutions at 80 C Even an overnight storage at 20 C may result in RNA degradation e When working with RNA always use gloves as hands are a major source of ribonuclease contamination e The high sensitivity of DNA amplification techniques makes the reactions susceptible to DNA contamination from previous amplification reactions potentially resulting in false positive results To minimize the risk of contamination avoid opening qPCR tubes after amplification e Laboratory workbenches and pipettes must be cleaned with bleach on a regularly scheduled basis e Use aerosol barrier pipette tips 6 PROTOCOL AT A GLANCE 2 uL per tube acy Y V V W VN v v e N d ce eS r SS amn gt gt Total RNA cDNA mix qPCR mix qPCR Interpretation cDNA 3 Add cDNA 4 Program qPCR 5 qPCR
4. Y axis on amplification plot with background subtracted fluorescence ABI 7500 ViiA7 Roche Light Cyclers 480 Fluorescence using Analysis option Abs Quant 2nd Derivative Max for All Samples Table 1 Examples of instrument nomenclature for the Y axis when the amplification plot shows background subtracted fluorescence Figure 4 Amplification plot from Agilent Mx3005P with X axis showing cycle number 20 000 gt and Y axis showing background subtracted fluorescence The threshold line is used to find the Ct value Ct is the intersection between the amplification curve and the threshold line dR The threshold line is set just above the base line 10 000 this example resulting Ct value of 24 4 Ct values are calculated automatically P the instrument The algorithms used 7 to calculate Ct values differ between eee 4 line QPCR instrument software packages The threshold line the amplification plot should be set manually to allow accurate determination threshold line should be set just above the background fluorescence so that the threshold line intersects the amplification curves when the fluorescence increases due to PCR amplification The threshold level should however be set sufficiently high to allow for fluctuations in background fluorescence and background fluorescence drift a linear increase in fluorescen
5. analysis denaturation synthesis to qPCR tubes Instrument 18 uL total Transfer to Spin 3x8 tube block 10 1 cycle Generate RNA 0 15 1 5 ug one cDNA tube seconds 95 C 15 min amplification v v v v plots and use 65 C 5 min Mix and spin 1 Remove and discard lids 40 cycles Interpretation v v v 95 C 30 sec Table O C 1 min 42 C 60 min Add 2 uL cDNA to each of 60 C 50 sec v v the 23 qPCR mixes 72 C 80 sec Spin 1 min 95 C 5 min v v Close tubes with new read 0 C 1 min optical lids FAM ROX CY5 v v Spin 1 min Spin 1 min Figure 2 Overview of HemaVision 28Q protocol HemaVision 28Q 7 DETAILED PROTOCOL 7 1 RNA Preparation Total RNA is extracted from blood or bone marrow cells A minimum of 0 15 ug total RNA with a minimum concentration of 8 ng uL RNA is needed for the subsequent steps Due to the inherent instability of RNA use patient samples as fresh as possible Blood samples can be stabilized in Paxgene Blood RNA tubes Cat No BD762165 Do NOT use Heparin for Stabilization of blood samples Total RNA should be stored at 80 C 7 2 Synthesis Incubate total RNA at 65 C for 5 minutes cool immediately on Spin tube for 1 minute to collect condensate Take one yellow capped tube with cDNA master mix from the HemaVision 28Q kit stored at 20 C Add 18 uL 0 15 1 5 ug of denatured total RNA to the cDNA master mix tube tube with yellow cap If necessary the RN
6. 003472 3 rri NG 029953 1 NM 006532 3 NM 005934 3 NG 029732 1 NG 023272 2 012889 2 NM 002126 4 NG_028279 1 NG_027720 1 NG_027813 1 NG_023258 1 lt NG 009299 1 016018 1 NG 028246 1 NG 023367 1 NG 012140 1 NG 029036 1 NM 000964 3 NG_030356 1 012126 1 NM 003189 2 NG 011443 1 a A 012889 2 Ul HemaVision 28Q DNA DIAGNOSTIC Runa HemaVision 28Q Availability amp questions Our team and distributors are always at hand to answer your questions Contact us to find your nearest HemaVision partner For more information contact DNA Diagnostic A S Voldbjergvej 16 8240 Risskov Denmark Tel 45 87 32 3050 Fax 45 87 32 3059 info dna diagnostic com www dna diagnostic com DNA Diagnostic A S previously named DNA Technology A S was established in 1992 DNA Diagnostic A S is an ISO 13485 2012 certified developer manufacturer and worldwide supplier of PCR based CE IVD marked in vitro diagnostic kits Cat No HV01 28Q USER MANUAL
7. A sample can be diluted with the provided RNase free H O tube with blue cap Mix and spin for 1 minute Incubate the cDNA reaction at 42 C for 60 minutes Inactivate Reverse Transcriptase enzyme by heating to 95 C for 5 minutes Cool the cDNA reaction tube on ice for 1 minute and spin it again for 1 minute to collect condensate 7 3 qPCR For each cDNA sample use one block of 3x8 tube qPCR master mix tubes Spin the 3x8 tubes for 10 second to collect qPCR master mix at the bottom of tubes Remove and discard the caps from one block of 3x8 tubes Be careful not to separate the three 8 tube strips from each other Also be careful to avoid cross contamination and spill of reaction mixes while removing the caps Add 2 uL cDNA reaction to each of the 23 tubes containing 23 uL qPCR master mix tube 1 23 Tube number 24 is empty and is used for orientation of the 3x8 tube block see figure 3 Figure 3 Positions of the 23 qPCR master mixes in each block of 3 x 8 tubes Tube number 24 is empty and can be used for orientation of the 24 tube block The hole in the upper right corner of each 8 tube strip depicted as marks the beginning of the strip and the centered hole marks the end of the strip The tube numbering refers to the tube numbering in the Interpretation table G0 Q 9 9 0 6 65 HemaVision 28Q e Close the 24 tubes with a fresh set of optical caps supplied with the kit Use gloves to avo
8. AM 3 FAM 3 FAM t 15 17 PML RARA bcr1 L form 1 FAM 1 FAM t 15 17 PML RARA bcr2 V form 4 FAM 3 ROX 3 ROX t 9 11 MLL MLLT3 een 11 FAM 14 FAM 14 FAM t 11 19 MLL MLLT1 13 FAM 14 ROX 14 ROX t 9 22 BCR ABL1 M bcr P210 20 ROX 20 ROX t 10 11 MLL MLLT10 22 FAM 22 FAM Fusion genes targeted with more than one pair of amplification primers may result in positive signals in more than one tube Use table above in combination with Interpretation Table to identify the breakpoint 14 HemaVision 28Q Q 2 C Q m 2 m 2 gt lt m 9 gt 2 2 C gt C C m 9 es O 2 2 lt UJ m AJ 9 Old Abbreviation HGNC Abbreviation Chromosome HGNC ID NCBI Accession MDS1 EVI1 HGNC 3498 N1 MN1 HGNC 7180 The HUGO Gene Nomenclature Committee HGNC approves a unique and meaningful name for every known human gene The table contains a list of all relevant genes for the HemaVision 28Q kit with the old gene name abbreviation and the corresponding HGNC abbreviation Furthermore the table contains the NCBI www ncbi nlm nih gov nucleotide accession numbers for the gene or transcript sequences targeted by HemaVision 28Q primers and probes 1 NG 012034 1 NG 027818 1 NM 005937 3 NM 001981 2 NM 006818 3 gt NM_001166693 1 NM_001207008 1 gt gt NM_004529 2 NM 005938 3 NG 011402 2 NG 009244 1 NG 023371 1 NG 009281 1 NM
9. KS AND LICENSES HemavVision is a registered trade mark of DNA Diagnostic A S Cy5 is a registered trade mark of GE Healthcare Mx3005P Stratagene and Agilent are registered trademarks of Agilent Technologies Inc Black Hole Quencher BHQ CAL Fluor and Quasar dye technology incorporated in this product are used under licensing agreement with Biosearch Technologies Inc and protected by U S and world wide patents issued or in application TABLE OF CONTENTS J PORPOSEOI TI uuu u 3 2 PRINCIPLE OF cic 4 COMPONENTS AND Dur UNTER Eae REUNIR ICE HET VEINS Ne Eee 5 oh i OUIBRED I QUIPMIENI E uu 5 3 PRECAUTION 6 PROTOCOLAT A GLANCE Uu 6 RE 7 Step 7 1 2 7 Step 7 2 cDNA Synt uuu u uuu E ua 7 E E00 uuu x u u unan 7 Step 7 4 Analysis amp 8 8 INTERPRETATION TABLE nya 12 9 HGNC GENE NAMES AND NCBI ACCESSION NUMBERS 14 HemaVision 28Q 1 PURPOSE OF THE TEST H
10. bes no 1 23 The IAC CY5 fluorescence should yield Ct values between 29 and 34 This is a control for functionality of the qPCR reaction If no CY5 signals are detected the test has failed either due to no addition of cDNA to the qPCR tube s incorrect instrument settings or evaporation from the qPCR tube s Repeat the test Due to competition for PCR constituents CY5 Ct value above 34 may occur when the tube is also positive for FAM or ROX fluorescence Check the Ct values for GUS 2 and ABL1 The Ct values should be below 28 in tube 7 GUS 25 in tube 8 B2M and 29 in tube 16 ABL1 This is a control for RNA extraction and cDNA synthesis have been functional GUS B2M ABL1 Ct values higher than the above mentioned values indicates that the quality of the RNA may be too low to generate a valid test result Repeat test with fresh RNA e Check for a FAM or ROX signal with Ct value below 35 in the translocation tubes 1 6 9 15 17 23 e Translocation tests with Ct values below 35 for and signals and amplification curves with exponential growth can be considered as true positive Use the Interpretation Table to identify the specific translocation e Ct values above cycle 35 for FAM and ROX signals may be the result of unspecific amplification false positive Repeat the test to confirm or reject the result When the second test is negative the sample is negative for the translocation observed in the first
11. c The FAM amplification plot for tube 4 shows an amplification curve with a Ct value below 35 Therefore the test is positive for t 15 17 q24 q21 PML RARA bcr3 S form See Interpretation Table d The ROX amplification plot shows no amplification for the tubes 1 23 Therefore the test is negative for ROX amplicons HemaVision 28Q 12 INTERPRETATION TABLE Tube Translocation Fusion Gene Fw primer Rev primer Flourochrome PML ex5 RARA ex3 CBFB ex3 MYH11 ex30 CBFB ex4 MYH11 ex34 RUNX1 ex6 RUNX1T1 ex3 ROX CY5 C C CY5 lt t 15 17 q24 q21 inv 16 p13 q22 inv 16 p13 q22 t 8 21 q22 q22 PML RARA bcr2 V CBFB MYH11 CBFB MYH11 RUNX1 RUNX1T1 PML RARA bcr2 V _ x CBFEMYHMD x RUNXI RUNXITI t 15 17 q24 q21 PML ex6a RARA ex3 CY5 t 9 11 p22 q23 MLL ex7 MLLT3 ex7 CY5 t 15 17 q24 q21 PML ex3 RARA ex3 AN CY5 t 9 11 p22 q23 MLL ex8 MLLT3 ex11 ROX CY5 t 11 19 q23 p13 1 MLL ex7 ELL ex3 CY5 t 16 21 p11 22 FUS ex6 ERG ex14 CY5 t 12 22 p13 q11 12 ETV6 2 1 ex2 e me t 6 9 p23 q34 DEK ex9 NUP214 ex19 ROX CY5 Reference gene GUS GUS ex11 GUS ex12 CY5 Reference gene B2M B2M ex2 B2M ex4 CY5 t 1 11 p32 q23 MLL EPS15 MLL ex8 9 EPS15 ex3 CY5 t 6 11 q27 q23 MLL MLLT4 MLL ex8 9 MLLT4 ex2 CY5 MIES t 1 19 q23 p13 TCF3 ex16 PBX1 ex3 t 12 21 p13 q22 ETV6 ex5 RUNX1 ex4b ROX
12. ce The threshold level can be set manually using the following the steps Cycles ABI 7500 7500 Fast ViiA 7 In the Analysis window choose Plot Type deltaRn vs Cycle and Graph Type Linear Unmark Auto Threshold and Auto Baseline Then click Show Threshold and with the mouse move the threshold line above the baseline Bio Rad CFX96 Click the Settings tab choose Baseline Setting and Baseline Subtracted Curve Fit Using the mouse adjust the threshold level above baseline Qiagen Rotor Gene Q Click Analysis choose the fluorescence to analyze click Linear Scale Slope Correct and Auto Scale In the right side panel under CT Calculation click the button next to Threshold and mark the threshold level on the amplification plot Roche LightCycler480 Choose Absolut Quantification and Fit Points Then click the tab Noise Band and click the Noiseband drop down button and choose Noiseband Fluoresc Use the mouse to set the Noise Band Agilient MxPro The automatic threshold level can be used Choose Adaptive Baseline Correction and Background based Threshold using cycle 6 11 and a sigma multiplier of 10 HemaVision 28Q Note The Ct values cannot be used for exact quantification of the fusion transcripts level since the fusion gene amplicons differ in length resulting in different PCR efficiencies e Check that a CY5 signal is present from the Internal Amplification Control IAC in tu
13. eaction mix e 12 extra set of optical caps for the qPCR tubes e Two tubes with RNase free H20 0 65 mL tubes with blue screw One user manual HemaVision 28Q is produced in 3 formats to suit various qPCR apparatus The qPCR mix can be supplied in 0 1 mL white low profile WLP 0 2 mL white regular profile WRP or 0 2 mL frosted regular profile FRP PCR tubes Adaptor plates can be supplied with the kit if the qPCR instrument requires a 96 tube plate format e g Roche LightCycler 480 and some ABI models The cDNA reaction tubes contain reverse transcriptase nucleotides buffer cDNA primers and IAC oligo template The qPCR tubes contain hot start Taq DNA polymerase nucleotides buffer and primers probes Tube no 1 6 9 15 17 23 contain primers and probes for both fusion genes and the IAC template Tube no 7 8 16 contain primers and probes for a reference gene and the IAC template Tube no 24 is empty To confirm the orientation of the PCR strips after qPCR analysis check for FAM amplification signals in tube no 7 8 16 and the absence of Cy5 amplification signal in tube no 24 The kit must be stored at 20 C Avoid thawing and freezing of the kit The qPCR tubes must be protected from strong light to avoid bleaching of the probes 4 REQUIRED EQUIPMENT e Centrifuge for 96 well plates or PCR 8 tube strips e Thermal Heating Block e A qPCR instrument with filters for FAM Abs 495 nm Em 520 nm ROX Abs
14. emaVision 28Q is a 4 hour CE IVD marked in vitro diagnostic test for qualitative screening of 28 chromosome translocations involved in chronic and acute leukemia The amplification plot allows professionals to react rapid and with precision in terms of treatment planning while profiting from the cost and labour effective screening process HemaVision 28Q provides a rapid screen for 28 translocations with more than 145 clinically relevant chromosomal breakpoints The test requires only limited hands on time and the procedure can be completed in 4 hours after RNA extraction HemaVision 28Q detects RNA transcripts from fusion genes using a RT qPCR procedure Alternative splice variants are also detected HemaVision 28Q detects the following 28 translocations t 3 5 q25 q34 NPM1 MLF1 t 9 9 q34 q34 SET NUP214 t 10 11 p12 q23 MLL MLLT10 HemaVision 28Q 2 PRINCIPLES OF THE TEST HemaVision 28Q is RT qPCR based assay for detection of leukemia associated fusion gene transcripts total RNA from whole blood or bone marrow samples Included in the kit are ready to use cDNA and qPCR Master Mixes cDNA is synthesized by adding purified RNA to the HemaVision 28Q ready to use cDNA reaction mix The resulting cDNA is added to 23 ready to use qPCR reaction tubes which contain specific PCR primers and probes for detection of fusion genes three reference genes and an internal amp
15. id grease and dust on the optical caps e Place the 3x8 tube block into the qPCR instrument Runthe gPCR reaction with the following program Step Time Temperature Cycles Comment 30 seconds 95 C DNA amplification 50 seconds 60 C Read fluorescence for FAM ROX and CY5 at the end of each 80 seconds 72 C extension step 7 3 1 Important notes for ABI 7500 and ViiA7 users e Ramp speed Use standard ramp speed not fast mode e Targets While defining the 3 targets FAM ROX and CY5 choose for all targets None as Quencher e Passive reference dye Select None instead of ROX as passive reference dye 7 3 2 Important notes for Roche LightCycler 480 users A Color Compensation CC file should be generated before the first run on the Light Cycler 480 and if the lamp has been replaced A CC kit with FAM ROX and CY5 dye is available from DNA Diagnostic qPCR software setup e Set the ramp speed to 2 2 C sec for annealing step and 4 4 C sec for the remaining steps e For the amplification step of the PCR program under Analysis Mode choose Quantification and Acquisition Mode Single e Filter choice Manually choose FAM Red 610 and CY5 7 4 Analysis and Interpretation Generate amplification plots showing e Thermal cycles on the X axis e Background subtracted fluorescence on the Y axis The exact name on the Y axis differs among qPCR instruments See table 1 HemaVision 28Q x Instrument
16. lification control IAC The qPCR is performed in a real time qPCR instrument with optical filters for detection of FAM ROX and CY5 fluorescence Amplification plots and Ct cycle at threshold values are used for identification of the translocation and fusion gene transcript using an easy to use interpretation table HemaVision 28Q detects fusion gene transcripts using specific PCR primers and probes The translocation specific primers bind to exons in the fusion gene enabling amplification of the region containing the breakpoint The primers are designed to detect multiple clinical relevant breakpoints and splice variants Figure 1 The qPCR master mix probes are dual labeled with a fluorophore and a quencher molecule at each end of the oligoes During the annealing step of the PCR the probe bind to the PCR products of the previous rounds Signals are generated in the subsequent elongation step as the 5 gt 3 exonuclease activity of the Taq polymerase enzyme degrades the hybridized probe This liberates the fluorophore from the quencher which thereby increases the fluorescence The fluorescence is measured at the end of the elongation step of every PCR cycle When the fluorescence for a translocation exceeds the threshold level before cycle 35 Ct 35 the test is positive The qPCR s are multiplexed by the use of FAM ROX and CY5 labeled probes This permits two translocation tests and the IAC to run in the same tube The identity of a po
17. sitive translocation test is easily deduced from the Interpretation Table As a control for the functionality of the qPCR reaction and for correct transfer of cDNA aliquots to the 23 qPCR reactions an Internal Amplification Control IAC is included in the cDNA reaction mix HemaVision 28Q also includes primers and probes for detection of the reference gene transcripts ABL 1 B2M and GUS Detection of the reference genes is a control for the integrity of the RNA sample and functionality of both cDNA and qPCR reactions BCR ABL1 m bcr M bcr u bcr Y Y gt Tube 13 gt Tube 14 gt Tube 15 Figure 1 Primers and probes in HemaVision 28Q are designed to detect multiple clinical relevant breakpoints In this example primers and probe for detection of t 9 22 fusion gene transcript BCR ABL1 are shown Primers are depicted as blue arrows FAM and ROX labeled probe as green and red lines and breakpoints as black triangles Exons are numbered for the fusion genes BCR and ABL1 The three breakpoint regions m bcr M bcr u bcr of BCR are detected with the primer and probe combination of qPCR tube 13 14 and 15 respectively HemaVision 28Q 3 KIT COMPONENTS AND STORAGE HemaVision 28Q contains reagents for 12 tests Included in HemaVision 28Q kit is the following components e 12 cDNA tubes with 42 uL reaction mix 0 65 mL tubes with yellow screw e 12 blocks of 24 qPCR tubes containing 23 uL qPCR r
18. test When the second test is also positive the sample may be positive for the corresponding translocation However we highly recommend using other diagnostic techniques to confirm the result from positive tests having Ct values above 35 10 HemaVision 28Q Flowchart for data interpretation z 11 HemaVision 28Q 22000 1 20000 a will TM 12000 Fluorescence nal seco 6000 and 2000 22000 1 20000 C 18000 16000 FAM 14000 12000 Fluorescence 10000 000 6000 4000 eo eeo oeoo 9 4 4 PML RARA bcr3 Fluorescence e e o o o o o o o o o 22000 20000 12000 16000 14000 12000 10000 2000 6000 4000 Figure 5 Example of amplification plots from a HemaVision 28Q test 8 B2M FAM f 7 GUS wen j o 2216 ABLA 4 B 10 20 Cycles 30 40 ROX 1 23 10 20 cycles 30 a The CY5 amplification plot shows all 23 IAC control curves are present and have Ct values near 32 This serves as a positive control for correct transfer of 2 uL cDNA to all 23 qPCR tubes and functionality of the qPCR reactions b The FAM amplification plot for the reference genes B2M GUS and ABL1 in tubes 8 7 16 all show curves with Ct below 25 28 and 29 respectively This is positive a control for both the RNA quality and the RT qPCR reactions were functional
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