A - Z of quantitative PCR:
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| Format: | Book |
|---|---|
| Language: | English |
| Published: |
La Jolla, Calif.
Internat. Univ. Line
2006
|
| Edition: | [Nachdr.] |
| Series: | IUL biotechnology series
5 |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XXIX, 882 S. Ill., graph. Darst. |
| ISBN: | 0963681788 9780963681782 |
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MARC
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| 245 | 1 | 0 | |a A - Z of quantitative PCR |c ed. by Stephen A. Bustin |
| 246 | 1 | 3 | |a A-Z of quantitative PCR |
| 250 | |a [Nachdr.] | ||
| 264 | 1 | |a La Jolla, Calif. |b Internat. Univ. Line |c 2006 | |
| 300 | |a XXIX, 882 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 1 | |a IUL biotechnology series |v 5 | |
| 650 | 7 | |a Genética quantitativa |2 larpcal | |
| 650 | 7 | |a Reação em cadeia por polimerase |2 larpcal | |
| 650 | 4 | |a Polymerase chain reaction | |
| 650 | 4 | |a Nucleic acids |x Analysis | |
| 650 | 4 | |a Quantitative genetics | |
| 650 | 0 | 7 | |a Nucleinbasen |0 (DE-588)4248593-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Quantitative Analyse |0 (DE-588)4125311-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Polymerase-Kettenreaktion |0 (DE-588)4256726-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Chemische Analyse |0 (DE-588)4009840-0 |2 gnd |9 rswk-swf |
| 689 | 0 | 0 | |a Nucleinbasen |0 (DE-588)4248593-9 |D s |
| 689 | 0 | 1 | |a Chemische Analyse |0 (DE-588)4009840-0 |D s |
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| 689 | 1 | 1 | |a Quantitative Analyse |0 (DE-588)4125311-5 |D s |
| 689 | 1 | |8 1\p |5 DE-604 | |
| 700 | 1 | |a Bustin, Stephen A. |e Sonstige |4 oth | |
| 830 | 0 | |a IUL biotechnology series |v 5 |w (DE-604)BV013752839 |9 5 | |
| 856 | 4 | 2 | |m Digitalisierung UB Regensburg |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016246714&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
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| 883 | 1 | |8 1\p |a cgwrk |d 20201028 |q DE-101 |u https://d-nb.info/provenance/plan#cgwrk | |
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| _version_ | 1804137272578146304 |
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| adam_text | 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. Bus
t
in
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
v¡
Contents
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.
RNAExtraction
............................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
.............................
Ill
3.6.
Conclusion
......................................112
Contents
vii
PART IL
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.
Preanałytical
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 Fiow 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
viii Contente
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
Contents
¡χ
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.
PNAProbe 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
x
Contents
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 Gycler®......................356
8.3. Outlook.........................................355
9. Basic RT-PCR
Considérations
.............................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.
RTProperties
..............................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
Contents xi
10.
The PCR Step
..........................................397
Stephen
Α.
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
xü
Contents
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 qPCRDoes 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
Contente
xiii
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 Oligonucleo-
tide 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
PARTin. 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
xiv
Contents
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,
ЋтоЛу
G
Graves, David A. Weaver, Jerry R. Bergman,
Martin Vondrecek, and Roland
C. Graf strom
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
Contents xv
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 KNA 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-cDNAII: 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
xvi Contents
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
Pinzarti,
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.
RNAExtraction
............................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
Contents xvii
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 Sty
................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.
DNAand RNAIsolation
...........................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
xviii Contents
23. Single Nucleotide
Polymorphism Detection with Fluorescent
MGB Eclipse Probe Systems
..............................717
Irina
A. Afemina,
Yevgeniy S. Belonsov, MarkMetcalf, Alan Mills,
Silvia Sanders, David K.
Walburger,
Walt Mahoney, and
Nicolaos
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
Contents xix
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
Alíele
Specific Primers and
Intercalation
...............................777
25.5.2.
Single-Tube Genotyping
.....................778
25.5.3.
Quantitative Genoryping 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
xx
Contente
APPENDICES
........................................797
Appendix
Al.
Useful Information
............................799
AU.
Sizes and Molecular Weights of Eukaryotic Genomic
DNA
and rRNAs
.................................801
Al
.2.
Nucleic Acids in Typical Human Cell
.................803
A1.3. Nucleotide Molecular Weights
.......................803
A
1.4.
Molecular Weights of Common Modifications
..........804
A
1.5.
Nucleic Acid Molecular Weight Conversions
...........804
Al
.6.
Nucleotide Absorbance Maxima and Molar Extinction
Coefficients
.....................................807
All Conversions
.....................................807
A1.8.
DNA
Conformations
..............................812
A1.9. Efficiency of PCR Reactions
........................812
ALIO.
Centrimgation
....................................813
Al.
11.
Splice Function
...................................813
Appendix A2. Glossary
.....................................815
Index
.....................................................835
|
| adam_txt |
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. Bus
t
in
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
v¡
Contents
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.
RNAExtraction
.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
.
Ill
3.6.
Conclusion
.112
Contents
vii
PART IL
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.
Preanałytical
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 Fiow 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
viii Contente
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
Contents
¡χ
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.
PNAProbe 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
x
Contents
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 Gycler®.356
8.3. Outlook.355
9. Basic RT-PCR
Considérations
.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.
RTProperties
.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
Contents xi
10.
The PCR Step
.397
Stephen
Α.
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
xü
Contents
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 qPCRDoes 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
Contente
xiii
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 Oligonucleo-
tide 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
PARTin. 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
xiv
Contents
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,
ЋтоЛу
G
Graves, David A. Weaver, Jerry R. Bergman,
Martin Vondrecek, and Roland
C. Graf strom
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
Contents xv
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 KNA 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-cDNAII: 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
xvi Contents
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
Pinzarti,
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.
RNAExtraction
.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
Contents xvii
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 Sty
.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.
DNAand RNAIsolation
.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
xviii Contents
23. Single Nucleotide
Polymorphism Detection with Fluorescent
MGB Eclipse Probe Systems
.717
Irina
A. Afemina,
Yevgeniy S. Belonsov, MarkMetcalf, Alan Mills,
Silvia Sanders, David K.
Walburger,
Walt Mahoney, and
Nicolaos
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
Contents xix
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
Alíele
Specific Primers and
Intercalation
.777
25.5.2.
Single-Tube Genotyping
.778
25.5.3.
Quantitative Genoryping 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
xx
Contente
APPENDICES
.797
Appendix
Al.
Useful Information
.799
AU.
Sizes and Molecular Weights of Eukaryotic Genomic
DNA
and rRNAs
.801
Al
.2.
Nucleic Acids in Typical Human Cell
.803
A1.3. Nucleotide Molecular Weights
.803
A
1.4.
Molecular Weights of Common Modifications
.804
A
1.5.
Nucleic Acid Molecular Weight Conversions
.804
Al
.6.
Nucleotide Absorbance Maxima and Molar Extinction
Coefficients
.807
All Conversions
.807
A1.8.
DNA
Conformations
.812
A1.9. Efficiency of PCR Reactions
.812
ALIO.
Centrimgation
.813
Al.
11.
Splice Function
.813
Appendix A2. Glossary
.815
Index
.835 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| building | Verbundindex |
| bvnumber | BV023043223 |
| callnumber-first | Q - Science |
| callnumber-label | QP606 |
| callnumber-raw | QP606.D46 |
| callnumber-search | QP606.D46 |
| callnumber-sort | QP 3606 D46 |
| callnumber-subject | QP - Physiology |
| classification_rvk | WC 4460 YV 3408 |
| ctrlnum | (OCoLC)246920884 (DE-599)BVBBV023043223 |
| dewey-full | 572.8/6 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.8/6 |
| dewey-search | 572.8/6 |
| dewey-sort | 3572.8 16 |
| dewey-tens | 570 - Biology |
| discipline | Biologie Medizin |
| discipline_str_mv | Biologie Medizin |
| edition | [Nachdr.] |
| format | Book |
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| id | DE-604.BV023043223 |
| illustrated | Illustrated |
| index_date | 2024-07-02T19:21:25Z |
| indexdate | 2024-07-09T21:09:40Z |
| institution | BVB |
| isbn | 0963681788 9780963681782 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016246714 |
| oclc_num | 246920884 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-20 DE-29T |
| owner_facet | DE-355 DE-BY-UBR DE-20 DE-29T |
| physical | XXIX, 882 S. Ill., graph. Darst. |
| publishDate | 2006 |
| publishDateSearch | 2006 |
| publishDateSort | 2006 |
| publisher | Internat. Univ. Line |
| record_format | marc |
| series | IUL biotechnology series |
| series2 | IUL biotechnology series |
| spelling | A - Z of quantitative PCR ed. by Stephen A. Bustin A-Z of quantitative PCR [Nachdr.] La Jolla, Calif. Internat. Univ. Line 2006 XXIX, 882 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier IUL biotechnology series 5 Genética quantitativa larpcal Reação em cadeia por polimerase larpcal Polymerase chain reaction Nucleic acids Analysis Quantitative genetics Nucleinbasen (DE-588)4248593-9 gnd rswk-swf Quantitative Analyse (DE-588)4125311-5 gnd rswk-swf Polymerase-Kettenreaktion (DE-588)4256726-9 gnd rswk-swf Chemische Analyse (DE-588)4009840-0 gnd rswk-swf Nucleinbasen (DE-588)4248593-9 s Chemische Analyse (DE-588)4009840-0 s DE-604 Polymerase-Kettenreaktion (DE-588)4256726-9 s Quantitative Analyse (DE-588)4125311-5 s 1\p DE-604 Bustin, Stephen A. Sonstige oth IUL biotechnology series 5 (DE-604)BV013752839 5 Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016246714&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | A - Z of quantitative PCR IUL biotechnology series Genética quantitativa larpcal Reação em cadeia por polimerase larpcal Polymerase chain reaction Nucleic acids Analysis Quantitative genetics Nucleinbasen (DE-588)4248593-9 gnd Quantitative Analyse (DE-588)4125311-5 gnd Polymerase-Kettenreaktion (DE-588)4256726-9 gnd Chemische Analyse (DE-588)4009840-0 gnd |
| subject_GND | (DE-588)4248593-9 (DE-588)4125311-5 (DE-588)4256726-9 (DE-588)4009840-0 |
| title | A - Z of quantitative PCR |
| title_alt | A-Z of quantitative PCR |
| title_auth | A - Z of quantitative PCR |
| title_exact_search | A - Z of quantitative PCR |
| title_exact_search_txtP | A - Z of quantitative PCR |
| title_full | A - Z of quantitative PCR ed. by Stephen A. Bustin |
| title_fullStr | A - Z of quantitative PCR ed. by Stephen A. Bustin |
| title_full_unstemmed | A - Z of quantitative PCR ed. by Stephen A. Bustin |
| title_short | A - Z of quantitative PCR |
| title_sort | a z of quantitative pcr |
| topic | Genética quantitativa larpcal Reação em cadeia por polimerase larpcal Polymerase chain reaction Nucleic acids Analysis Quantitative genetics Nucleinbasen (DE-588)4248593-9 gnd Quantitative Analyse (DE-588)4125311-5 gnd Polymerase-Kettenreaktion (DE-588)4256726-9 gnd Chemische Analyse (DE-588)4009840-0 gnd |
| topic_facet | Genética quantitativa Reação em cadeia por polimerase Polymerase chain reaction Nucleic acids Analysis Quantitative genetics Nucleinbasen Quantitative Analyse Polymerase-Kettenreaktion Chemische Analyse |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016246714&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV013752839 |
| work_keys_str_mv | AT bustinstephena azofquantitativepcr |