Contents

Contributors xiii

Abbreviations xv

Preface xvii

Glossary xix

Links to software and web resources cited xxv

1 An introduction to real-time PCR 1

Gregory L. Shipley

1.1 Introduction 1

1.2 A brief history of nucleic acid detection and quantification 1

1.3 Real-time quantitative PCR - a definition 3

1.4 Practical and theoretical principles underlying real-time PCR 3

1.5 Real-time PCR instrumentation - an overview 12

1.6 Detection chemistries used in real-time PCR 16

1.7 Performing a real-time RT-PCR experiment 25

1.8 What lies ahead 29 References 30 Protocol 1.1 32 Protocol 1.2 33 Protocol 1.3 35 Protocol 1.4 36 Protocol 1.5 37

2 Data analysis and reporting 39

Pamela Scott Adams

2.1 Introduction 39

2.2 Standard curves 40

2.3 Preliminary assay analysis 45

2.3.1 Amplification curves 46

2.3.2 Baseline 47

2.3.3 Threshold 50

2.3.4 Proper controls 52

2.3.5 Experimental samples 56

2.3.6 Quantifying data 57

2.4 Data reporting and statistics 59 References 61

3 Relative quantification 63

Michael W. Pfaffl

3.1 Introduction 63

3.2 Relative quantification: The quantification is relative to what? 64

3.3 Normalization 65

3.4 Mathematical models 66

3.5 Real-time qPCR amplification efficiency 68

3.6 Determination of the amplification rate 69

3.6.1 Dilution method 70

3.6.2 Fluorescence increase in exponential phase 71

3.6.3 Sigmoidal or logistic curve fit 72

3.6.4 Efficiency calculation in the exponential phase using 73 multiple models

3.7 What is the right crossing point to determine? 75

3.8 Relative quantification data analysis and software applications 76

3.8.1 LightCycler® Relative Quantification Software 76

3.8.4 qBASE 78

3.8.5 SoFAR 79

3.8.6 DART-PCR 79

3.9 Conclusion 79 References 80

4 Normalization 83

Jim Huggett, Keertan Dheda and Stephen A. Bustin

4.1 Introduction 83

4.2 General error and directional shift 83

4.3 Methods of normalization 84

4.3.1 Sample size 84

4.3.2 Total RNA amount 86

4.3.3 Quantification of cDNA 87

4.3.4 PCR dependent reference 87

4.4 Conclusion 90 References 90

5 High-throughput primer and probe design 93

Xiaowei Wang and Brian Seed

5.1 Primer and probe design guidelines 93

5.1.1 Primer specificity 93

5.1.2 Primer length 93

5.1.3 Primer GC content 93

5.1.4 Primer 3' end stability 94

5.1.5 Primer sequence complexity 94

5.1.6 Primer melting temperature 94

5.1.7 Primer location in the sequence 95

5.1.8 Amplicon size 96

5.1.9 Cross-exon boundary 96

5.1.10 Primer and template sequence secondary structures 96

5.1.11 TaqMan® probe design 96

5.1.12 Molecular beacon probe design 97

5.2 PrimerBank - an online real-time PCR primer database 97

5.2.1 Primer design algorithm 97

5.2.2 PrimerBank 100

5.2.3 Experimental validation of the primer design 101

5.3 Experimental protocol using PrimerBank primers 101

5.3.1 Reverse transcription (RT) 101

5.3.2 Real-time PCR 102

5.3.3 Troubleshooting 103

5.3.4 Little or no PCR product 103

5.3.5 Poor PCR amplification efficiency 103

5.3.6 Primer dimer 103

5.3.7 Multiple bands on gel or multiple peaks in the melting curve 104

5.3.8 Non-specific amplification 104

5.4 Web resources about primer and probe design 104

5.4.1 Real-time PCR primer and probe databases 104

5.4.2 Primer and probe design tools 104

5.4.3 Other useful web sites 105 References 105

6 Quantitative analysis of ocular gene expression 107

Stuart N. Peirson

Summary 107

6.1 Introduction 107

6.1.1 Gene expression in the eye 107

6.1.2 Problems associated with ocular gene expression 108

6.2 Relative quantification 112

6.2.1 The R0 method 112

6.2.2 Kinetic approaches to qPCR 114

6.2.3 Accurate normalization 116

6.3 Assay considerations 118

6.4 Conclusions 124 Acknowledgments 124 References 124

7 Quantitative gene expression by real-time PCR: a complete protocol 127

Thomas D. Schmittgen

7.1 Introduction 127

7.2 Materials 127

7.2.1 Reagents and consumables 127

7.2.2 Equipment 128

7.3 Procedure 128

7.3.1 Sample preparation 128

7.3.2 Isolation of RNA from cultured cells or blood 128

7.3.3 Isolation of RNA from whole tissue 129

7.3.4 RNA quantification 130

7.3.5 DNase treatment 130

7.3.6 cDNA synthesis 130

7.3.8 Primer design 131

7.3.9 Real-time PCR 132

7.3.10 Data analysis 133

7.4 Troubleshooting 135

7.5 Critical steps 136

7.6 Comments 136 References 137

8 Real-time PCR using SYBR® Green 139

Frederique Ponchel

8.1 Introduction 139

8.2 SYBR® Green chemistry 139

8.3 Primer design 140 8.3.1 Step by step primer design: P-actin for a cDNA quantification 141

assay

8.4 Primer optimization 142

8.4.1 Absolute quantification of gene expression 142

8.4.2 Relative quantification of gene expression 142

8.4.3 Relative quantification of different gene modifications 145 (amplification, deletion, rearrangement, translocation)

8.5 Melting curve analysis 146

8.6 Quantification of gene modification 146

8.6.1 DNA quantification 147

8.6.2 Gene amplification 147

8.6.3 Gene deletion 147

8.6.4 Gene rearrangement 148

8.6.5 Gene copy number 149

8.7 RNA quantification 149

8.7.1 RNA extraction 149

8.7.2 cDNA preparation 149

8.7.3 Reference gene validation 150

8.7.4 Splice variants and splicing machinery 150

8.7.5 Promoter switch 150

8.8 Allelic discrimination 152

8.9 Chromatin immunoprecipitation 152

8.10 Conclusion 152 References 153

9 High-resolution melting analysis for scanning and genotyping 155

Virginie Dujols, Noriko Kusukawa, Jason T. McKinney, Steven F. Dobrowolsky and Carl T. Wittwer

9.1 Introduction 155

9.2 High-resolution instrumentation 156

9.2.1 The HR-1TM instrument 156

9.2.2 The LightScanner™ instrument 156

9.2.3 The LightCycler® 480 instrument 157

9.3 Saturating dyes 157 9.3.1 LCGreen™ dyes 158

9.4 Mutation scanning 159

9.4.1 PCR protocols for scanning 159

9.4.2 Principles of scanning by melting 160

9.4.3 Software tools for heterozygote identification 161

9.4.4 Scanning for homozygous variants 163

9.5 Amplicon genotyping 163

9.6 Unlabeled probe genotyping 164

9.6.1 PCR protocols for unlabeled probe genotyping 164

9.6.2 Instrumentation for unlabeled probe genotyping 165

9.6.3 Simultaneous genotyping and scanning 165

9.7 Simplification of genotyping and mutation scanning 165 References 167

10 Quantitative analyses of DNA methylation 171

Lin Zhou and James (Jianming) Tang

10.1 Introduction 171

10.2 MDR1 (ABCB1, Gene ID 5243) as a primary target locus 172

10.3 Primer design 173

10.4 Data evaluation: I. Assay-to-assay variability 174

10.5 Data evaluation: II. MDR1 CpG methylation as quantified by qPCR 174

10.6 Expanded analyses 176 Acknowledgments 176 References 176 Protocol 10.1 179

11 Mitochondrial DNA analysis 183

Steve E. Durham and Patrick F. Chinnery

11.1 Introduction 183

11.2 Mitochondrial genetics 183

11.2.1 mtDNA mutations 185

11.2.2 mtDNA copy number and heteroplasmy 185

11.2.3 The threshold effect 185

11.2.4 Mutation rate of mtDNA 186

11.2.5 Mitochondrial DNA, ageing and disease 186

11.3 Mitochondrial DNA analysis by real-time PCR 189

11.3.1 Detection method 189

11.3.2 Oligonucleotide fluorescent probes 189

11.3.3 DNA binding dyes 190

11.3.4 Considerations when designing a mtDNA real-time assay 191

11.4 Discussion 201 References 203

12 Real-time immuno-PCR 205

Kristina Lind and Mikael Kubista

12.1 Introduction 205

12.1.1 Immunoassays 205

12.1.2 Immuno-PCR 205

12.2 Assemblages for real-time immuno-PCR 206

12.2.1 Attaching capture antibody 207

12.2.2 Labeling detection antibody with DNA 207

12.3 Real-time immuno-PCR details 207

12.3.1 Reaction containers and instruments 207

12.3.2 DNA-label 208

12.3.3 Blocking agents 208

12.3.4 Controls 208

12.3.5 Optimizing concentrations 209 References 210 Protocol 12.1 211

13 Clinical microbiology 219

Burcu Cakilci and Mehmet Gunduz

13.1 Introduction 219

13.1.1 Importance of detection and quantification in microbiology 219

13.1.2 From traditional methods to real-time PCR in microbiology 219

13.2 Real-time PCR studies in microbiology 220

13.2.1 Basics for microbial quantitation 220

13.2.2 Bacteria 221

13.2.3 Fungi and parasites 224 References 225 Protocol 13.1 229

14 Clinical virology 231

David M. Whiley and Theo P. Sloots

14.1 Introduction 231

14.2 Qualitative real-time PCR for viral disease 232 14.2.1 Sequence variation and assay performance 232

14.3 Virus typing using sequence-specific probes 236

14.3.1 Hybridization probes 236

14.3.2 Additional comments 239

14.4 Quantification of viral load 239

14.4.1 The use of an internal control in clinical molecular virology 239

14.4.2 Impact of target sequence variation on qPCR 240

14.4.3 Additional comments 241

14.5 Conclusions 242 References 242 Protocol 14.1 244 Protocol 14.2 246 Protocol 14.3 248 Protocol 14.4 250 Troubleshooting guide 14.1 251 Troubleshooting guide 14.2 254 Troubleshooting guide 14.3 256 Troubleshooting guide 14.4 257

15 Solid organ transplant monitoring 259

Omaima M. Sabek

15.1 Introduction 259

15.2 Real-time quantitative PCR 260

15.3 RNA normalization 260

15.4 Immunologic monitoring in solid organ transplantation 261

15.5 Pharmacogenetics in solid organ transplantation 261

15.6 Cytokine gene polymorphism analysis 262 15.6.1 Recipient and donor gene polymorphisms 263

15.6.2 Ethnicity and cytokine gene polymorphism 263

15.7 Viral infection in transplant patients 264

References 265

Protocol 15.1 270

16 Real-time PCR applications in hematology 277

Anne M. Sproul

16.1 Specimens 278

16.2 Specimen quality 278

16.3 Template preparation 278

16.4 DNA isolation 278

16.5 PCR inhibition 278

16.6 RNA isolation 279

16.7 cDNA synthesis 279

16.8 Relative versus absolute quantitation 280

16.9 Control genes for MRD in leukemia 281

16.10 Controls for real-time PCR 282

16.11 Assay design 283

16.12 Laboratory precautions 283

16.13 PCR reaction set-up 284

16.14 Interpretation and quantitation 284

16.15 Sensitivity 285

16.16 Targets for detecting MRD 285

16.16.1 Fusion transcripts 285

16.16.2 Rearrangements of immunoglobulin/ 287 TCR genes in lymphoid neoplasia

References 288

Protocol 16.1 290

Protocol 16.2 298

0 0

Post a comment