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1 January 2009 Rotor-Gene Q Sample & Assay Technologies
2 Rotor-Gene Q Rotor-Gene Q Run File PCR Auto-Gain Optimization Analysis menu Analysis Quantitation Two Standard Curve C T Delta delta C T relative Quantitation Melt curve analysis Comparative Quantitation Allelic Discrimination Scatter Graph Analysis High Resolution Melt PCR HRM 5-7 Rotor-Gene Q 01/2009
3 1 1.1 Rotor-Gene Q Virtual Mode Rotor-Gene Q File Set up 1.2 Rotor-Gene Q 1. Rotor-Gene Q USB 2. USB 1.3 Rotor-Gene Q 1. Windows 2. Rotor-Gene Q 3. Rotor-Gene Q Well Rotor 72-Well Rotor Rotor-Gene Q 01/
4 72-Well Rotor Rotor-Gene Q 01/2009
5 2 Rotor-Gene Q 2.1 New SYBR Green I 3 PCR 2.1 Run File File New Advanced Perform Last Run Empty Run Three Step with Melt Two Step HRM Other Runs Instrument Maintenance 3 PCR 2 PCR PCR HRM HRM Optical Temperature Verification (OTV) Rotor-Gene Q 01/
6 2.2 Locking Ring Attached 36-Well Rotor 2.3 Operator Notes Report 2-2 Rotor-Gene Q 01/2009
7 2.4 PCR Edit Profile Rotor-Gene Q 01/
8 2.6 PCR + Insert after Insert before Remove 2.7 Not Acquiring Acquiring to Cycling A SYBR Green I Green Acquired channels OK > 2-4 Rotor-Gene Q 01/2009
9 2.8 DNA 95 T m 99 1 /step 2.9 Auto-Gain Optimization 2-3 Auto-Gain Optimization PCR Gain SYBR Green Optimize Acquiring Green 1 3 Perform Optimization Before 1st Acquisition Tube Position 1 1 NTC Rotor-Gene Q 01/
10 Auto-Gain Optimization Target Sample Range Gain Target Sample Range SYBR Green I TaqMan Probe Target Sample Rang 1 3 Target Sample Rang Start Run Run File 2-6 Rotor-Gene Q 01/2009
11 3 Samples Standard, Unknown etc Edit Reset Default Gradient New icon Open icon Save icon Copy icon Paste icon Excel icon Sample Types Excel None NTC Negative Control Positive Control Unknown Standard No template control Rotor-Gene Q 01/
12 Page Standard Page Page 1 1 Page 1 New Page 2 Page 2 Page < > Page 1 Page 2 Page Page Page Standard Page Page 1 Page 2 Page 1 Standard Page 3-2 Rotor-Gene Q 01/2009
13 4 4.1 Settings Progress Profile Temp. Samples Analysis Reports Arrange 4.2 Analysis menu Analysis Analysis Show Show Hide Remove Analysis Show Rotor-Gene Q 01/
14 4.2.2 Quantitation Quantitation Amplification Curve Dynamic Tube Normalization 5 6 Dynamic Tube Normalization Starting Point 1 Starting Point ON Noise Slope Correction ON 10 Outlier Removal Adjust Scale Maximum Minimum Auto-Scale Default Scale Adjust Scale Auto-Scale 4-2 Rotor-Gene Q 01/2009
15 Log Linear Log Linear Scale Linear Log. Scale C T Calculation C T Threshold Threshold Threshold Threshold Threshold Threshold Auto-Find Threshold Upper Lower Threshold Threshold Standard R 1.0 Upper Lower Threshold Standard Unknown Efficiency R^2 value (correlation coefficient) PCR Standard R^ Rotor-Gene Q 01/
16 R value (square root of correlation coefficient) M and B R^2 M B Y = Mx + B Results Excel %Var Rep. C T Rep. C T Std. Dev. Rep. C T 95% C.I. Rep. Calc. Conc Rep. Calc. Conc. 95% C.I. % %Var=Abs(Calculated/Given 1) C T C T C T 95% C T 95% C T 4-4 Rotor-Gene Q 01/2009
17 4.2.3 Two Standard Curve Two Standard Curve Page Page Analysis 2 Std Curves (Rel.) New Analysis Show 2. Rotor-Gene Q 01/
18 3. Gene of Interest Standard Curve Normaliser Standard Curve 4. Calibrator Calibrator 1 Calibrator Defined C T Delta delta C T relative Quantitation C T C T C T C T C T C T C T C T C T C T C T * * Analysis of relative gene expression data using real-time quantitative PCR and the 2^[ -delta delta C(T) ] Method. Livak KJ & Schmittgen TD. Methods 2001 Dec;25(4): Rotor-Gene Q 01/2009
19 1. Analysis Delta Delta C T Relative Quantitation New Analysis Show 2. Validation Run Performed Gene of Interest Quantitation Normaliser Quantitation Rotor-Gene Q 01/
20 3. Calibrator Calibrator 1 1 Calibrator Defined Melt curve analysis PCR T m T m 1. Analysis Melt Show 2. Threshold Threshold Peak Calling Threshold Threshold Threshold Threshold line Threshold line 4-8 Rotor-Gene Q 01/2009
21 Bin Bin Bin Peak Bins New Bin Bin Genotype Bin Genotype Bin Rotor-Gene Q 01/
22 4.2.6 Comparative Quantitation Comparative Quantitation 1. Analysis Other Comparative Quantitation 2. DNA Second Derivative Maximum Take off Point Take off Point Second Derivative Maximum Calibrator Replicate Second Derivative Maximum Take Off Point Comparative Quantitation PCR 2nd Derivative Maximum Take Off Point DNA exponential phase Comparative Concentration Take Off Point Amplirication 4-10 Rotor-Gene Q 01/2009
23 4.2.7 Allelic Discrimination Allelic Discrimination 2 PCR 1. Analysis Other Allelic Discrimination Shift Ctrl Show 2. Allelic Discrimination Threshold line Discrimination Threshold Threshold 3. Genotype Scatter Graph Analysis Scatter Graph Analysis Allelic Discrimination 2 1. Analysis Other Scatter Analysis Shift Ctrl Show 2. Genotypes 3. Rotor-Gene Q 01/
24 High Resolution Melt 5 High Resolution Melt 5.1 Software setup 1. File New Advanced High Resolution Melt High Resolution Melt 2. Locking Ring Attached 72-Well Rotor 5-1 Rotor-Gene Q 01/2009
25 High Resolution Melt 3. Operator Notes Report 4. Edit Profile Rotor-Gene Q 01/
26 High Resolution Melt 6. PCR 7. Green Acquired channels OK 5-3 Rotor-Gene Q 01/2009
27 High Resolution Melt 8. HRM 10 HRM Gain Gain Optimization 70 HRM PCR Gain Optimize Acquiring Green 1 3 Perform Optimization Before 1st Acquisition 10. Start Run Run File Rotor-Gene Q 01/
28 High Resolution Melt 11. Standard Unknown OK 5-5 Rotor-Gene Q 01/2009
29 High Resolution Melt 5.2 PCR HRM PCR HRM PCR PCR Quantitation analysis C T 30 HRM C T 30 Analysis Mode Comparative Quantitation 1.4 Comparative Quantitation Comparative Quantitation Amplification 100 Amplification 2 Analysis Mode Melt 2 PCR PCR Rotor-Gene Q 01/
30 High Resolution Melt 5.3 HRM HRM Normalized melt plot Difference plot Normalized melt plot Homozygotes Heterozygotes Difference plot Difference Melt HRM HRM Normalization 1. Analysis HRM Show Analysis HRM Other High Resolution Melt 2. HRM Normalized 3 HRM Normalization region Normalized Normalization region 5-7 Rotor-Gene Q 01/2009
31 High Resolution Melt 3. Normalized Difference plot 5 4. Difference plot Genotype Genotype Genotype Control 5. Difference plot Normalized Normalized graph Difference graph Genotype Rotor-Gene Q 01/
32 High Resolution Melt 6. Genotype Confidence Percentage Confidence Percentage Confidence 90 variation 5-9 Rotor-Gene Q 01/2009
33 Memo Rotor-Gene Q 01/2009
34 Memo Rotor-Gene Q 01/2009
35 Memo Rotor-Gene Q 01/2009
36 Trademarks: QIAGEN (QIAGEN Group), Rotor-Gene (Corbett Research); SYBR (Molecular Probes, Inc.); TaqMan (Roche Group). QIAGEN 2009 QIAGEN, all rights reserved Forefront Tower II Tel: FAX: /2009 Sample & Assay Technologies
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