A-Z of Quantitative PCR

$119.95

IUL Biotechnology Series, 5
by Stephen A. Bustin (Editor)
Edition: First

Print: Third

Book Details:

  • Series: IUL Biotechnology Series
  • Volume: 5
  • Binding: Hardcover 
  • Pages: 912
  • Dimensions (in inches): 1.75 x 9.50 x 6.50
  • Publisher: International University Line 
  • Publication Date: August, 2004
  • ISBN: 0-9636817-8-8
  • Price: $109.95

Look Inside This Book

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

 

A-Z of Quantitative PCR

$119.95