Verfügbarkeit: A - Z of quantitative PCR

A - Z of quantitative PCR:

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Format:	Buch
Sprache:	English
Veröffentlicht:	La Jolla, Calif. Internat. Univ. Line 2006
Ausgabe:	[Nachdr.]
Schriftenreihe:	IUL biotechnology series 5
Schlagworte:	Genética quantitativa Reação em cadeia por polimerase Polymerase chain reaction Nucleic acids > Analysis Quantitative genetics Nucleinbasen Quantitative Analyse Polymerase-Kettenreaktion Chemische Analyse
Online-Zugang:	Inhaltsverzeichnis
Beschreibung:	XXIX, 882 S. Ill., graph. Darst.
ISBN:	0963681788 9780963681782

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650		4	\|a Nucleic acids \|x Analysis
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Datensatz im Suchindex

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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
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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
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