January 2009 Rotor-Gene Q Sample & Assay Technologies
1 1-1 1.1 1-1 1.2 Rotor-Gene Q 1-1 1.3 Rotor-Gene Q 1-1 1.4 1-1 2 2-1 2.1 Run File 2-1 2.2 2-2 2.3 2-2 2.4 PCR 2-3 2.5 2-3 2.6 2-4 2.7 2-4 2.8 2-5 2.9 Auto-Gain Optimization 2-5 2.10 2-6 3 3-1 4 4-1 4.1 4-1 4.2 Analysis menu 4-1 4.2.1 Analysis 4-1 4.2.2 Quantitation 4-2 4.2.3 Two Standard Curve 4-5 4.2.4 C T Delta delta C T relative Quantitation 4-6 4.2.5 Melt curve analysis 4-8 4.2.6 Comparative Quantitation 4-10 4.2.7 Allelic Discrimination 4-11 4.2.8 Scatter Graph Analysis 4-11 5 High Resolution Melt 5-1 5.1 5-1 5.2 PCR 5-6 5.3 HRM 5-7 Rotor-Gene Q 01/2009
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 1.4 36-Well Rotor 72-Well Rotor Rotor-Gene Q 01/2009 1-1
72-Well Rotor 1. 2. 3. 1. 2. 1-2 Rotor-Gene Q 01/2009
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 01/2009 2-1
2.2 Locking Ring Attached 36-Well Rotor 2.3 Operator Notes Report 2-2 Rotor-Gene Q 01/2009
2.4 PCR Edit Profile 2.5 10 Rotor-Gene Q 01/2009 2-3
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
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 01/2009 2-5
Auto-Gain Optimization Target Sample Range Gain Target Sample Range SYBR Green I TaqMan Probe Target Sample Rang 1 3 Target Sample Rang 5 10 2.10 Start Run Run File 2-6 Rotor-Gene Q 01/2009
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 01/2009 3-1
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
4 4.1 Settings Progress Profile Temp. Samples Analysis Reports Arrange 4.2 Analysis menu 4.2.1 Analysis Analysis Show Show Hide Remove Analysis Show Rotor-Gene Q 01/2009 4-1
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
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^2 0.99 Rotor-Gene Q 01/2009 4-3
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
4.2.3 Two Standard Curve Two Standard Curve Page Page 3-2 1. Analysis 2 Std Curves (Rel.) New Analysis Show 2. Rotor-Gene Q 01/2009 4-5
3. Gene of Interest Standard Curve Normaliser Standard Curve 4. Calibrator Calibrator 1 Calibrator Defined 4.2.4 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): 402-408. 4-6 Rotor-Gene Q 01/2009
1. Analysis Delta Delta C T Relative Quantitation New Analysis Show 2. Validation Run Performed Gene of Interest Quantitation Normaliser Quantitation Rotor-Gene Q 01/2009 4-7
3. Calibrator Calibrator 1 1 Calibrator Defined 4.2.5 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
Bin Bin Bin Peak Bins New Bin Bin Genotype Bin Genotype Bin Rotor-Gene Q 01/2009 4-9
4.2.6 Comparative Quantitation Comparative Quantitation 1. Analysis Other Comparative Quantitation 2. DNA Second Derivative Maximum 100 20 Take off Point Take off Point Second Derivative Maximum Calibrator Replicate Second Derivative Maximum Take Off Point Comparative Quantitation PCR 2nd Derivative Maximum 100 20 Take Off Point DNA exponential phase Comparative Concentration Take Off Point Amplirication 4-10 Rotor-Gene Q 01/2009
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 4.2.8 Scatter Graph Analysis Scatter Graph Analysis Allelic Discrimination 2 1. Analysis Other Scatter Analysis Shift Ctrl Show 2. Genotypes 3. Rotor-Gene Q 01/2009 4-11
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
High Resolution Melt 3. Operator Notes Report 4. Edit Profile 5. 10 Rotor-Gene Q 01/2009 5-2
High Resolution Melt 6. PCR 7. Green Acquired channels OK 5-3 Rotor-Gene Q 01/2009
High Resolution Melt 8. HRM 10 HRM Gain Gain Optimization 70 HRM 70 70 9. PCR Gain Optimize Acquiring Green 1 3 Perform Optimization Before 1st Acquisition 10. Start Run Run File Rotor-Gene Q 01/2009 5-4
High Resolution Melt 11. Standard Unknown OK 5-5 Rotor-Gene Q 01/2009
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 01/2009 5-6
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
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 01/2009 5-8
High Resolution Melt 6. Genotype Confidence Percentage Confidence Percentage 90 90 Confidence 90 variation 5-9 Rotor-Gene Q 01/2009
Memo Rotor-Gene Q 01/2009
Memo Rotor-Gene Q 01/2009
Memo Rotor-Gene Q 01/2009
Trademarks: QIAGEN (QIAGEN Group), Rotor-Gene (Corbett Research); SYBR (Molecular Probes, Inc.); TaqMan (Roche Group). QIAGEN 2009 QIAGEN, all rights reserved. www.qiagen.co.jp 104-0054 3-13-1 Forefront Tower II Tel:03-6890-7300 FAX: 03-5547-0818 E-mail:techservice-jp@qiagen.com 2301515 01/2009 Sample & Assay Technologies