A-Z of Quantitative PCR
edited by
Stephen A. Bustin


Contents
Preface     xxi
List of Contributors     xxiii
Acronims and Abbreviations    xxvii
Part I.  OVERVIEWS     1
1.    Quantification of Nucleic Acids by PCR     3
Stephen A. Bustin
       1.1.    Introduction     5
                 1.1.1.    PCR Characteristics     6
       1.2.    Conventional Quantitative PCR     8
                 1.2.1.    Concepts     10
                 1.2.2.    Limitations     12
                 1.2.3.    Alternatives      13
       1.3.    Real-Time Quantitative PCR     15
                 1.3.1.    Uses     16
                 1.3.2.    Microdissection     19
                 1.3.3.    Limitations     22
                 1.3.4.    PCR     22
                 1.3.5.    RT-PCR     23
       1.4.    Outlook     26
       1.5.    Conclusion     29
2.    Real-Time RT-PCR: What Lies Beneath the Surface     47
Jonathan M. Phillips
       2.1.    Introduction     49
       2.2.    What is RT-PCR?     50
                 2.2.1.    Reverse Transcription and RT Enzymes     52
                 2.2.2.    What is Quantitative RT-PCR?     57
                 2.2.3.    Real-Time RT-PCR     58
                 2.2.4.    Reaction Controls (IPCs)     58
                 2.2.5.    Reporter Technologies     60
       2.3.    Things That Influence RT-PCR     61
                 2.3.1.    Why Commercial Kits?     62
                 2.3.2.    Divalent Metal Concentration     64
                 2.3.3.    Primer Concentration     65

                 2.3.4.    Probe Concentration     66
                 2.3.5.    Reverse Transcription Conditions     67
       2.4.    Synthetic Molecules     70
                 2.4.1.    Substituted Primers and Probes     70
                 2.4.2.    Synthetic RNA Controls     71
       2.5.    A Word about DNA Polymerases     73
                 2.5.1.    DNA Dependent DNA Polymerases     73
                 2.5.2.    RNA Dependent DNA Polymerases     74
       2.6.    Tips and Tricks     75
                 2.6.1.    Probes     75
                 2.6.2.    The Right Enzyme for the Job     77
       2.7.    Buffers     78
       2.8.    Concluding Remarks     78
3.    Quantification Strategies in Real-Time PCR     87
Michael W. Pfaffl
       3.1.    Introduction     89
       3.2.    Markers of a Successful Real-Time RT-PCR Assay     90
                 3.2.1.    RNA Extraction     90
                 3.2.2.    Reverse Transcription     91
                 3.2.3.    Comparison of Real-Time RT-PCR with Classical Endpoint Detection Method     93
                 3.2.4.    Chemistry Developments for Real-Time RT-PCR     94
                 3.2.5.    Real-Time RT-PCR Platforms     94
                 3.2.6.    Quantification Strategies in Kinetic RT-PCR     95
                 3.2.7.    Advantages and Disadvantages of External Standards     100
                 3.2.8.    Real-Time PCR Amplification Efficiency     102
                 3.2.9.    Data Evaluation     105
       3.3.    Automation of the Quantification Procedure     106
       3.4.    Normalization     108
       3.5.    Statistical Comparison     111
       3.6.    Conclusion     112
PART II.  BASICS     121
4.    Good Laboratory Practice!     123
Stephen A. Bustin and Tania Nolan
       4.1.    Introduction     125
       4.2.    General Precautions     126
                  4.2.1.    Phenol     127
                               Emergency procedures in case of skin contact     128
                 4.2.2.    Liquid Nitrogen (N2)     129
                 4.2.3.    Waste Disposal     130
       4.3.    Equipment     131
                 4.3.1.    Electrophoresis     131
                 4.3.2.    Freezer     131
                 4.3.3.    UV Transilluminators     131
                 4.3.4.    Micropipettes     132
                 4.3.5.    Gloves     134
                 4.3.6.    Eye Protection     135
                 4.3.7.    Legal Information     136
5.    Template Handling, Preparation, and Quantification     141
Stephen A. Bustin and Tania Nolan
       5.1.    Introduction     143
                 5.1.1.    General Precautions     144
       5.2.    DNA     146
                 5.2.1.    Preanalytical Steps     146
                 5.2.2.    Sample Collection     150
                 5.2.3.    Disruption     151
                 5.2.4.    Purification     154
                 5.2.5.    Long-Term Storage     159
       5.3.    RNA     159
                 5.3.1.    Preanalytical Steps     160
                 5.3.2.    General Considerations     161
                 5.3.3.    Tissue Handling and Storage     163
                 5.3.4.    Disruption/Homogenization     165
                 5.3.5.    RNA Extraction     173
                 5.3.6.    Simultaneous DNA Extraction     180
                 5.3.7.    DNA Contamination     182
                 5.3.8.    Preparation of RNA from Flow Cytometrically Sorted Cells     183
                 5.3.9.    Extraction from Formalin-Fixed and Paraffin-Embedded Biopsies     184
                 5.3.10.    Specialized Expression Analysis     187
       5.4.    Quantification of Nucleic Acids     188
                 5.4.1.    Absorbance Spectrometry     188
                 5.4.2.    Fluorescence     190
                 5.4.3.    Purity     190
                 5.4.4.    Quantification of RNA     191
6.    Chemistries     215
Stephen A. Bustin and Tania Nolan
       6.1.    Introduction     217
       6.2.    Fluorescence     221
                 6.2.1.    Fluorophores     222
                 6.2.2.    Quenchers     226
       6.3.    Nonspecific Chemistries     228
                 6.3.1.    DNA Intercalators     228
                 6.3.2.    Advantages     229
                 6.3.3.    Disadvantages     231
                 6.3.4.    Quencher-Labeled Primer (I)     234
                 6.3.5.    Quencher-Labeled Primer (II)     234
                 6.3.6.    LUX™ Primers     235
                 6.3.7.    Amplifluor™     236
       6.4.    Specific Chemistries     239
                 6.4.1.    Advantages     240
                 6.4.1.    Disadvantages     240
       6.5.    Linear Probes     241
                 6.5.1.    ResonSense® and Angler® Probes     241
                 6.5.2.    HyBeacons™     242
                 6.5.3.    Light-up Probes     243
                 6.5.4.    Hydrolysis (TaqMan®) Probes     244
                 6.5.5.    Lanthanide Probes     246
                 6.5.6.    Hybridization Probes     249
                 6.5.7.    Eclipse™     249
                 6.5.8.    Displacement Hybridization/Complex Probe     250
       6.6.    Structured Probes     251
                 6.6.1.    Molecular Beacons     253
                 6.6.2.    Scorpions™     259
                 6.63.    Cyclicons™     261
       6.7.    Future Technology     263
                 6.7.1.    Nanoparticle Probes     263
                 6.7.2.    Conjugated Polymers And Peptide Nucleic Acid Probes     263
7.    Primers and Probes     279
Stephen A. Bustin and Tania Nolan
       7.1.    Introduction     281
                 7.1.1.    Hybridization     283
       7.2.    Probe Design     288
       7.3.    Hydrolysis Probes     290
                 7.3.1.    Gene Expression Analysis     290
                 7.3.2.    SNP/Mutation Analysis     292
       7.4.    Hybridization Probes     293
                 7.4.1.    Gene Expression Analysis     293
                 7.4.2.    SNP/Mutation Analysis     294
       7.5.    Molecular Beacons     294
                 7.5.1.    Gene Expression Analysis     295
                 7.5.2.    SNP/Mutation Analysis     296
       7.6.    Scorpions™     296
                 7.6.1.    Gene Expression Analysis     297
                 7.6.2.    SNP/Mutation Analysis     299
       7.7.    Probe Storage     299
       7.8.    Primer Design     299
       7.9.    Amplifluor™ Primers     303
       7.10.    LUX™ Primers     304
       7.11.    Oligonucleotide Purification     305
       7.12.    Recommended Storage Conditions     307
       7.13.    Example of Primer Design     308
       7.14.    Nucleic Acid Analogues     311
                   7.14.1.    Peptide Nucleic Acids (PNA)     313
                   7.14.2.    PNA Probe Characteristics     315
                   7.14.3.    Locked Nucleic Acids LNA™     317
                   7.14.4.    Modified Bases: Super A™, G™, and T™     318
                   7.14.5.    Minor Groove Binding Probes     319
8.    Instrumentation     329
Stephen A. Bustin and Tania Nolan
        8.1.    Introduction     331
                  8.1.1.    The Principle     332
                  8.1.2.    Excitation Source     333
                  8.1.3.    Filters     335
                  8.1.4.    Photodetectors     337
                  8.1.5.    Sensitivity     339
                  8.1.6.    Dynamic Range     340
                  8.1.7.    Linearity     340
       8.2.    Real-Time Instruments     341
                 8.2.1.    ABI Prism®     345
                 8.2.2.    Bio-Rad Instruments     346
                 8.2.3.    Stratagene’s Instruments     348
                 8.2.4.    Corbett Research Rotor-Gene RG-3000     350
                 8.2.5.    Roche Applied Science     353
                 8.2.6.    Techne Quantica     355
                 8.2.7.    Cepheid Smart Cycler®     356
       8.3.    Outlook     355
9.    Basic RT-PCR Considerations     359
Stephen A. Bustin and Tania Nolan
       9.1.    Introduction     361
       9.2.    Total RNA vs. mRNA     364
       9.3.    cDNA Priming     364
                 9.3.1.    Random Primers     365
                 9.3.2.    Oligo-dT     366
                 9.3.3.    Target-Specific Primers     366
       9.4.    Choice of Enzyme     366
                 9.4.1.    RT Properties     367
                 9.4.2.    AMV-RT     370
                 9.4.3.    MMLV-RT     371
                 9.4.4.    DNA-Dependent DNA Polymerases     372
                 9.4.5.    Omniscript/Sensiscript     372
       9.5.    RT-PCR     372
                 9.5.1.    Two-Enzyme Procedures: Separate RT and PCR Enzymes     373
                 9.5.2.    Single RT and PCR Enzyme     374
                 9.5.3.    Problems with RT     375
       9.6.    One-Enzyme/One-Tube RT-PCR Protocol     376
                 9.6.1.    Preparations     376
                 9.6.2.    Primers and Probes     376
                 9.6.3.    RT-PCR Enzyme     377
                 9.6.4.    RT-PCR Solutions     377
                 9.6.5.    Preparation of Master Mix     377
                 9.6.6.    Preparation of Standard Curve     378
                 9.6.7.    Template Reaction     380
                 9.6.8.    Troubleshooting     381
       9.7.    Two-Enzyme/Two-Tube RT-PCR Protocol     382
                 9.7.1.    RT-PCR Enzymes     382
                 9.7.2.    RT-PCR Solutions     382
                 9.7.3.    Preparation of Master Mix     382
                 9.7.4.    Preparation of Standard Curve     383
                 9.7.5.    Unknown Template Reaction     385
                 9.7.6.    Troubleshooting     386
10.    The PCR Step     397
Stephen A. Bustin and Tania Nolan
         10.1.    Introduction     399
         10.2.    Choice of Enzyme     400
         10.3.    Thermostable DNA Polymerases     401
                     10.3.1.    Fidelity     406
                     10.3.2.    Processivity and Elongation Rates     406
                     10.3.3.    Thermostability     407
                     10.3.4.    Robustness     407
       10.4.    To UNG or not to UNG     410
       10.5.    Hot Start PCR     411
       10.6.    PCR Assay Components     413
                  10.6.1.    Enzyme Concentration     413
                  10.6.2.    Mg2+ Concentration     414
                  10.6.3.    Primers     414
                  10.6.4.    dNTPs     415
                  10.6.5.    Template     416
                  10.6.6.    Inhibition of PCR by RT Components     417
                  10.6.7.    Water     417
       10.7.    Reaction Conditions     417
                   10.7.1.    Denaturation Temperature     418
                   10.7.2.    Annealing Temperature     418
                   10.7.3.    Polymerization Temperature     418
                   10.7.4.    Reaction Times     419
                   10.7.5.    Multiplexing     419
                   10.7.6.    Additives     419
       10.8.    PCR Protocols for Popular Assays     422

                   10.8.1.    Preparations     423
                   10.8.2.    Double Stranded DNA Binding Dye Assays     424
                   10.8.3.    Hydrolysis (TaqMan) Probe Reaction     426
                   10.8.4.    Molecular Beacon Melting Curve to Test Beacon and Scorpion Assays     429
                   10.8.5.    Molecular Beacon/Scorpion Reaction     430
       10.9.    General Troubleshooting     431
11.    Data Analysis and Interpretation     439
Stephen A. Bustin and Tania Nolan
        11.1.    Introduction     441
        11.2.    Precision, Accuracy, and Relevance     442
        11.3.    Quantitative Principles     444
        11.4.    Effect of Initial Copy Numbers     446
        11.5.    Monte Carlo Effect     447
        11.6.    Amplification Efficiency     448
        11.7.    Relative, Comparative or Absolute Quantification     449
        11.8.    Absolute Quantification     450
        11.9.    Standard Curves     451
                    11.9.1.    Recombinant DNA     454
                    11.9.2.    Genomic DNA     455
                    11.9.3.    SP6 or T7-Transcribed RNA     456
                    11.9.4.    Universal RNA     456
                    11.9.5.    Sense-Strand Oligonucleotides     457
        11.10.    Relative Quantification     458
        11.11.    Normalization     460
                      11.11.1.    Tissue Culture     461
                      11.11.2.    Nucleated Blood Cells (NBC)     462
                      11.11.3.    Solid Tissue Biopsies     462
                      11.11.4.    Cell Number     463
                      11.11.5.    Total RNA     463
                      11.11.6.    DNA     464
                      11.11.7.    rRNA     464
        11.12.    Reference Genes (Housekeeping Genes)     465
        11.13.    Basic Statistics     467
                      11.13.1.    Data Presentation     469
                      11.13.2.    Mean and Median     469
                      11.13.3.    Standard Deviation     470
                      11.13.4.    Plots     470
                      11.13.5.    Relative (Receiver) Operating Characteristics     471
                      11.13.6.    Probability     473
                      11.13.7.    Parametric and Nonparametric Tests     475
        11.14.    Conclusion     481
12.    The qPCR Does Not Work?     493
Stephen A. Bustin and Tania Nolan
         12.1.    Introduction     495
         12.2.    Problem: What Is a Perfect Amplification Plot?     496
         12.3.    Problem: Too Much Target     498
                     12.9.1.    Solution     499
         12.4.    Problem: Amplification Plot Is not Exponential     499
                    12.4.1.    Solution     500
         12.5.    Problem: Duplicates Give Widely Differing Cts     500
                     12.5.1.    Solution     502
         12.6.    Problem: No Amplification Plots     502
                     12.6.1.    Solution     502
         12.7.    Problem: The Probe Does not Work!     506
                     12.7.1.    Solution     510
         12.8.    Problem: The Data Plots Are Very Jagged     511
                     12.8.1.    Solution     511
         12.9.    Problem: The Amplification Plot for the Standard Curve Looks Great BUT……………    512
                     12.9.1.    ……..The Gradient of the Standard Curve Is Greater Than -3.3     514
                     12.9.2.    ……..The Standards Aren’t Diluting!     515
                     12.9.3.    ……..Using SYBR Green the Gradient of the Standard Curve Is Less Than -3.3     517
                     12.9.4.    ……..Using a Sequence Specific Oligonucleotide Detection System the Gradient of the
                                                       Standard Curve Is Less Than -3.3     518
                     12.10.    Problem: The Amplification Plots Are Strange Wave Shapes     521
                                   12.10.1.  Solution     522
                     12.11.    Problem: The Amplification Plot Goes Up, Down and All Around     523
                                   12.11.1.  Solution     523
PART III.  SPECIFIC APPLICATIONS     525
13.    Getting Started—The Basics of Setting up a qPCR Assay     527
Tania Nolan
         13.1.    Introduction     529
         13.2.    Optimization     531
         13.3.    Primer and Probe Optimization Protocol     532
         13.4.    Optimization of Primers Concentration Using SYBR Green I     534
         13.5.    SYBR Green 1 Optimization Data Analysis     535
         13.6.    Examination of the Melting Curve     535
         13.7.    Optimization of Primer Concentration Using Fluorescent Probes     537
         13.8.    Molecular Beacon Melting Curve     537
         13.9.    Primer Optimization Reactions in Duplicate     538
         13.10.    Primer Optimization Data Analysis     539
         13.11.    Optimization of Probe Concentration     539
         13.12.    Probe Optimization Data Analysis     542
         13.13.    Testing the Efficiency of Reactions Using a Standard Curve     542
14.    Use of Standardized Mixtures of Internal Standards in Quantitative RT-PCR to Ensure 
         Quality Control and Develop a Standardized Gene Expression Database     545

James C. Willey, Erin L. Crawford, Charles A. Knight, Kristy A. Warner, Cheryl R. Motten, 
Elizabeth Herness Peters, Robert J. Zahorchak, Timothy G. Graves, David A. Weaver, 
Jerry R. Bergman, Martin Vondrecek, and Roland C. Grafstrom

         14.1.    Introduction     547
                     14.1.1.    Controls Required for RT-PCR to Be Quantitative     548
                     14.1.2.    Control for Variation in Loading of Sample into PCR Reaction     548
                     14.1.3.    Control for Variation in Amplification Efficiency     552
                     14.1.4.    Control for Cycle-to-Cycle Variation in Amplification     552
                     14.1.5.    Control for Gene-to-Gene Variation in Amplification Efficiency     552
                     14.1.6.    Control for Sample-to-Sample Variation in Amplification Efficiency     553
                     14.1.7.    Control for Reaction-to-Reaction Variation in Amplification Efficiency     554
                     14.1.8.    Schematic Comparison of StaRT-PCR to Real-Time     556
         14.2.    Materials     559
         14.3.    Methods     560
                     14.3.1.    RNA Extraction and Reverse Transcription     560
                     14.3.2.    Synthesis and Cloning of Competitive Templates     560
                     14.3.3.    Preparation of Standardized Mixtures of Internal Standards     562
         14.4.    StaRT-PCR     563
                    14.4.1.    Step-by-Step Description of StaRT-PCR Method     564
         14.5.    The Standardized Expression Measurement Center     570
         14.6.    Technology Incorporated by the SEM Center     571
                     14.6.1.    Automated Preparation of StaRT-PCR Reactions     571
                     14.6.2.    Electrophoretic Separation of StaRT-PCR Products     572

                     14.6.3.    Design of High-Throughput StaRT-PCR Experiments     572
15.    Standardization of qPCR and qRT-PCR Assays     577
Reinhold Mueller, Gothami Padmabandu, and Roger H. Taylor
         15.1.    Introduction     579
         15.2.    Platforms     581
                     15.2.1.    Validation of Instrument Specification     581
         15.3.    Detection Chemistries     586
         15.4.    Conclusion     588
16.    Extraction of Total RNA from Formalin-Fixed Paraffin-Embedded Tissue     591
Fraser Lewis and Nicola J. Maughan
         16.1.    Introduction     593
         16.2.    Extraction of RNA from Clinical Specimens     594
         16.3.    Effect of Fixation     595
         16.4.    Extraction of total RNA from Formalin-Fixed, Paraffin-Embedded Tissue     596
         16.5.    Use of RNase Inhibitors     597
         16.6.    Protocol for the Extraction of total RNA from Formalin-Fixed, Paraffin-Embedded Tissue     598
                     16.6.1.    Method     598
         16.7.    Reverse Transcription of Total RNA from Paraffin Sections     600
                    16.7.1.    Method     600
         16.8.    Design of Real-Time PCR Assays     601
17.    Cells-to-cDNA II:  RT-PCR without RNA Isolation     605
Quoc Hoang and Brittan L. Pasloske
         17.1.    Introduction     607
         17.2.    Materials     609
                     17.2.1.    Materials Supplied with Cells-to-cDNA II     609
                     17.2.2.    Materials for Real-Time PCR     609
                     17.2.3.    Heating Sources     610
        17.3.    Method     610
                    17.3.1.    Lysis and DNase I Treatment     610
                    17.3.2.    Reverse Transcription     611
                    17.3.3.    Real-Time PCR     611
                    17.3.4.    Data Analysis     612
         17.4.    Notes     613
18.    Optimization of Single and Multiplex Real-Time PCR     619
Marni Brisson, Shannon Hall, R. Keith Hamby, Robert Park, and Hilary K Srere
         18.1.    Introduction     621
                     18.1.1.    Why Multiplex?     622
         18.2.    Getting Started—Proper Laboratory Technique     623
                     18.2.1.    Avoiding Contamination     623
                     18.2.2.    Improving Reliability     624
         18.3.    Designing Probes for Multiplexing     624
                     18.3.1.    Types of Probes     624
                     18.3.2.    Reporters and Quenchers     624
                     18.3.3.    Analyzing Probe Quality     626
         18.4.    Standard Curves     627
                     18.4.1.    Interpreting Standard Curves     627
                     18.4.2.    Proper Use of Standards     628
         18.5.    Optimizing Individual Reactions before Multiplexing     630
                     18.5.1.    Definition of Efficiency     630
                     18.5.2.    Designing Primers for Maximum Amplification Efficiency     631
                     18.5.3.    Designing Primers for Maximum Specificity     632
                     18.5.4.    Equalizing Amplification Efficiencies     635
         18.6.    Optimization of Multiplex Reactions     636
                     18.6.1.    Comparing Individual and Multiplexed Reactions     636
                     18.6.2.    Optimizing Reaction Conditions     636
         18.7.    Summary     640
19.    Evaluation of Basic Fibroblast Growth Factor mRNA Levels in Breast Cancer     643
Pamela Pinzani, Carmela Tricarico, Lisa Simi, Mario Pazzagli, and Claudio Orlando
         19.1.    Introduction     645
         19.2.    Materials and Methods     647
                     19.2.1.    Cancer Samples     647
                     19.2.2.    Materials     647
                     19.2.3.    Sample Preparation     648
                     19.2.4.    Quantitative Evaluation of bFGF mRNA Expression     648
                     19.2.5.    Statistical Analysis     648
         19.3.    Results     649
                     19.3.1.    Intra-Assay and Inter-Assay Variability     649
                     19.3.2.    Quantification of bFGF and VEGF mRNA Levels     649
                     19.3.3.    Clinicopathologic Characteristics     650
         19.4.    Discussion     653
20.    Detection of “Tissue-Specific” mRNA in the Blood and Lymph Nodes of 
         Patients without Colorectal Cancer     657

Stephen A. Bustin and Sina Dorudi
         20.1.    Introduction     659
         20.2.    Materials and Methods     661
                     20.2.1.    Patients and Controls     661
                     20.2.2.    Tumors and Lymph Nodes     661
                     20.2.3.    RNA Extraction     662
                     20.2.4.    Primers and Probes     663
                     20.2.5.    RT-PCR Reactions     663
                     20.2.6.    Quantification     664
                     20.2.7.    Normalization     664
                     20.2.8.    Quality Standards     665
         20.3.    Results     665
                     20.3.1.    ck20 mRNA in Colorectal Cancers     665
                     20.3.2.    ck20 mRNA  in the Peripheral Blood of Patients     665
                     20.3.3.    ck20 mRNA in the Peripheral Blood of Healthy Volunteers     667
                     20.3.4.    ck20 Expression in Lymph Nodes     667
                     20.3.5.    ck20 Expression in Other Human Tissues     667
         20.4.    Discussion     668
21.    Optimized Real-Time RT-PCR for Quantitative Measurements of DNA and RNA 
          in Single Embryos and Their Blastomeres     675

Cristina Hartshorn, John E. Rice, and Lawrence J. Wangh
         21.1.    Introduction     677
         21.2.    Key Features of Real-Time RT-PCR     680
         21.3.    Primer Design     681
         21.4.    Avoidance of the HMG Box within Sry     681
         21.5.    Amplicon Selection and Verification     682
         21.6.    Molecular Beacons Design     684
         21.7.    Multiplex Optimization     686
         21.8.    Blastomere Isolation     688
         21.9.    DNA and RNA Isolation     691
         21.10.    Reverse Transcription     694
         21.11.    Real-time PCR and Quantification of Genomic DNA and cDNA Templates in Single Embryos     696
         21.12.    Real-time PCR and Quantification of Genomic DNA and cDNA Templates in Single Blastomeres     698
22.    Single Cell Global RT and Quantitative Real-Time PCR     703
Ged Brady and Tania Nolan
         22.1.    Introduction     705
         22.2.    PolyAPCR Overview     706
         22.3.    Ensuring Ratio of RNAs in Is Equal to Ratio of cDNAs out     707
         22.4.    Why Carry out Single Cell Analysis?     707
         22.5.    Picking the “Right” Single Cell     709
         22.6.    Experimental Details of PolyAPCR     710
                     22.6.1.    Global Amplification of cDNA to Copy All Polyadenylated RNAs (PolyAPCR)     710
                     22.6.2.    Preparation of Gene Specific Quantity Standard Series     712
                     22.6.3.    TaqMan™ Real-Time Quantitative PCR to Quantify Specific Gene Expression     712
23.    Single Nucleotide Polymorphism Detection with Fluorescent MGB Eclipse Probe Systems     717
Irina A. Afonina, Yevgeniy S. Belousov, Mark Metcalf, Alan Mills, Silvia Sanders, David K. Walburger, 
Walt Mahoney, and Nicolaas M. J. Vermeulen

         23.1.    Introduction     719
         23.2.    General Discussion     721
         23.3.    Materials     723
                     23.3.1.    Preparation of Nucleic Acids     723
                     23.3.2.    Primers and Probes     724
                     23.3.3.    Amplification Enzyme     724
                     23.3.4.    Amplification Solutions     724
         23.4.    Method     724
                     23.4.1.    Amplification     724
                     23.4.2.    Melting Curve Analysis     725
         23.5.    Instruments     726
         23.6.    Data Interpretation     726
                     23.6.1.    Rotor-Gene     726
                     23.6.2.    Other Instruments     726
         23.7.    Notes     727
         23.8.    Summary     730
24.    Genotyping Using MGB-Hydrolysis Probes     733
Jane Theaker
         24.1.    Introduction     735
                     24.1.1.    Improved Chemistries     736
                     24.1.2.    Dark Quenchers     736
                     24.1.3.    Single-Tube Genotyping Assay Design Recommendations     737
         24.2.    Evaluation of a Single-Tube Genotyping Assay     738
         24.3.    Troubleshooting a Genotyping Assay     739
                     24.3.1.    Problem: No Signal or Poor Signal     739
                     24.3.2.    Problem: Probe Cross-Hybridization     741
                     24.3.3.    Problem: Spectral Crosstalk     742
         24.4.    The Transition from Real-Time to Endpoint Genotyping Assay     744
         24.5.    General Practical Points and Hints     745
                     24.5.1.    Plasticware and its Compatibility with Hardware     745
                     24.5.2.    ROX Including Baseline Drift     746
         24.6.    Software     750
                     24.6.1.    MFold     750
                     24.6.2.    HyTher™ Server 1.0    750
                     24.6.3.    Primer Express® Software     751
                     24.6.4.    Oligo Primer Analysis Software     751
                     24.6.5.    Beacon Designer 2.1     752
                     24.6.6.    Microsoft Excel     752
                     24.6.7.    JMP Version 5.1     752
         24.7.    Reagents and Buffers     752
                     24.7.1.    Alternative Suppliers of Reagents     753
                     24.7.2.    Formulate Your Own Reagents     754
         24.8.    Melting Curves     755
                     24.8.1.    Types of Melting Curves     755
                     24.8.2.    Performing a Pre-PCR Melting Curve     756
                     24.8.3.    Post-PCR Melting Curves     760
         24.9.    A Useful Protocol to Quantify Total Human DNA Based on Detection of the APO B Gene     763
                     24.9.1.    Primer and Probe Sequences     763
25.    Scorpions Primers for Real-Time Genotyping and Quantitative Genotyping on Pooled DNA     767
David M. Whitcombe, Paul Ravetto, AntonyHalsall, and Nicola Thelwell
         25.1.    Introduction     769
         25.2.    Genotyping     770
         25.3.    Scorpions     771
                     25.3.1.    Structure and Mechanism     771
                     25.3.2.    Benefits of the Scorpions Mechanism     772
        25.4.    Methods     773
                    25.4.1.    Design of ARMS Allele-Specific Primers     774
                    25.4.2.    Design and Synthesis of Scorpions     774
         25.5.    Examples     777
                     25.5.1.    Genotyping with Allele Specific Primers and Intercalation     777

                     25.5.2.    Single-Tube Genotyping     778
                    25.5.3.    Quantitative Genotyping of Pooled Samples     779
         25.6.    Conclusions     780
26.    Simultaneous Detection and Sub-Typing of Human Papillomavirus in the Cervix Using 
         Real-Time Quantitative PCR     783

Rashmi Seth, Tania Nolan, Triona Davey, John Rippin, Li Guo, and David Jenkins
         26.1.    Introduction     785
         26.2.    PolyAPCR Overview     788
         26.3.    Results     790
         26.4.    Conclusion     793
APPENDICES     797
Appendix A1.   Useful Information     799
         A1.1.    Sizes and Molecular Weights of Eukaryotic Genomic DNA and rRNAs     801
         A1.2.    Nucleic Acids in Typical Human Cell     803
         A1.3.    Nucleotide Molecular Weights     803
         A1.4.    Molecular Weights of Common Modifications     804
         A1.5.    Nucleic Acid Molecular Weight Conversions     804
         A1.6.    Nucleotide Absorbance Maxima and Molar Extinction Coefficients     807
         A1.7.    Conversions     807
         A1.8.    DNA Conformations     812
         A1.9.    Efficiency of PCR Reactions     812
         A1.10.    Centrifugation     813
         A1.11.    Splice Function     813
Appendix A2.   Glossary     815
Index     835


Preface

This is not just a cook book for real-time quantitative PCR (qPCR). Admittedly, there are lots of recipes from distinguished contributors and I have attempted to collect, sift through and rationalize the vast amount of information that is available on this subject. And yes, this book was conceived as a comprehensive hands-on manual to allow both the novice researcher and the expert to set up and carry out qPCR assays from scratch. However, this book also sets out to explain as many features of qPCR as possible, provide alternative viewpoints and methods and, perhaps most importantly, aims to stimulate the researcher into generating, interpreting and publishing data that are reproducible, reliable, and biologically meaningful. 
     The first of the reviews in part I describes the background to quantification using PCR-based assays (S. A. Bustin), the second one provides a fascinating insight into the numerous factors that influence a successful PCR experiment (J. M. Phillips), and the third review discusses in detail the principles underlying real-time quantification (M. Pfaffl). Part II forms the core of this book and presents a detailed dissection of every one of the steps involved in conducting a qPCR experiment. Its emphasis is on providing explanations at each critical step in the PCR assay, starting from sample collection and ending with the interpretation of the quantitative result. Tried and tested sample protocols are included for the main chemistries, together with a “getting started” section for the complete novice and an extensive troubleshooting section which details and explains problems encountered during everyday qPCR assays. 
     The third part of the book provides an alternative viewpoint and protocol for mRNA quantification (J. C. Willey et al.), specific guidelines for the standardization of qPCR assays (R. Mueller et al.) and protocols designed to optimize the extraction of RNA from formalin-fixed tissue (F. Lewis and N. J. Maughan), perform RT-PCR assays without the need to isolate the RNA in the first place (Q. Hoang and B. Pasloske) and detailed instructions on how to optimize multiplex PCR assays (H. K. Srere et al.). The remaining chapters are concerned with specific applications of real-time PCR assays in breast (P. Pinzani et al.) and colorectal (S. A. Bustin and S. Dorudi) cancer, quantification in single cells (C. Hartshorn et al.; G. Brady and T. Nolan), and SNP analyses (I. A. Afonina et al. and J. Theaker). Each chapter contains an abundance of practical hints and reveals technical information that the authors have acquired as part of their extensive exposure to this technique.
     The very nature of the technology means that new chemistries, protocols, and instruments come and go. Any book would struggle to keep up-to-date with such developments. However, by emphasizing and describing the very basic steps that must be right and providing step-by-step guidance on how to achieve reproducible results and interpret them correctly, this book will remain topical. My hope is that this book will contribute to taking quantitative PCR forward to a new stage of use as a standard, reliable, and useful molecular technique.
     I am grateful to my numerous friends and contacts at ABI, Ambion, Biorad, Corbett Research, DXS Genotyping, Oswell, Roche, Stratagene, and Quanta Biotech that keep me supplied with a constant stream of useful information, a lot of which has found a home in this book. I would like to acknowledge financial support from Bowel and Cancer Research.

Stephen A. Bustin
London, June 2004