Quantitation of mRNA by Polymerase Chain Reaction: Nonradioactive PCR Methods

摘要: I Theoretical and Methodical Prerequisites for Using PCR to Quantitate Nucleic Acids.- 1.1 General Aspects Chances of Acid Quantitation by PCR.- 1.1.1 Theory Template Amplification 1.1.1.1 Mathematical Description the Reaction.- 1.1.1.2 The Plateau Phase 1.1.2 Experimental Approaches mRNA.- 1.1.2.1 Quantitative External Standards.- Titration Analysis.- Kinetic 1.1.2.2 Internal Endogenous mRNA as Control.- Competitive Exogenous Added RNA/DNA 1.1.3 Sensitivity Reproducibility RT-PCR Assays.- 1.1.4 Methods Detection Products.- 1.1.5 Avoidance Contamination.- 1.2 Design Suitable Primers Competitor Fragments 1.2.1 Primer Selection.- 1.2.2 Construction Synthetic 1.2.2.1 Genes Serving Multifunctional Standards (Multistandards).- 1.2.2.2 Competitors Site-Directed Mutagenesis.- Subsequent Cloning Strategies.- Products Controls.- 1.2.3 What Should be Standard Choice?.- References.- 1.3 Short DNA In Vitro Transcription Generate RNA 1.3.1 Background.- 1.3.2 Procedures.- 1.3.2.1 T/A Procedure.- Ligation.- Transformation.- 1.3.2.2 Minipreparation Plasmid DNA.- 1.3.2.3 Digestion Isolated with Restriction Endonucleases.- 1.3.2.4 Cloned T7 Polymerase.- 1.4 Direct Non-lsotopic Sequencing or 1.4.1 Aspects.- 1.4.2 1.4.2.1 Cyclic Double Stranded 1.4.2.2 Solid-Phase Single 2Conventional Techniques 2.1 Isolation 2.1.1 2.1.2 Precautions in Isolation.- 2.1.3 2.1.4 2.1.4.1 Total-RNA RNAzol B.- 2.1.4.2 Purification Dynabeads Oligo (dT)25.- 2.1.5 Purified 2.1.6 Storage RNA.- 2.2 Synthesis cDNA.- 2.2.1 2.2.2 2.2.2.1 Reverse Transcriptase Reaction AMV-RT.- 2.2.2.2 RT rTth 2.3 Qualitative RT-PCR: Synthesized mdr-1 2.3.1 2.3.2 2.3.2.1 Polymerase Chain (PCR), Basic Protocol.- 2.3.2.2 Analysis Agarose Gel Electrophoresis.- 2.3.2.3 Digesting Enzymes.- 2.3.2.4 Polyacryl Amide Electrophoresis Silver Staining Fragments.- 2.4 Single-Tube RT-PCR.- 2.4.1 2.4.2 2.4.2.1 Single-Stranded cDNA Taq 2.4.2.2 mdrl 2.5 Nonradioactive Determination a DIG-Labeled Probe (Dot Blot).- 2.5.1 2.5.2 2.5.2.1 Preparation Dot Blots.- 2.5.2.2 Hybridization Blots Probe.- 2.5.2.3 DNA-DNA Hybrids.- 2.6 Northern Blot Probes.- 2.6.1 Principle Application Hybridization.- 2.6.2 Probes PCR-Generated 2.6.2.1 2.6.2.2 2.6.2.3 DIG-Labeling Random Priming.- 2.6.2.4 Estimating Yield 2.6.3 2.6.3.1 Through Denaturing Gels Containing Formaldehyde.- 2.6.3.2 Capillary Transfer Denatured Nylon Membrane.- 2.6.4 2.6.5 Immunological DNA-RNA Colorimetric Detection.- Chemiluminscent 2.6.6 2.6.6.1 Evaluation Size.- 2.6.6.2 Semiquantitative Steady-State Levels Specific 2.6.7 Stripping Reprobing After 3 Protocols Measurement Acids 3.1 ELOSA Technique 3.1.1 Technique.- 3.1.2 mRNAs PCR-ELOSA 3.1.3 3.1.3.1 PCR-ELOSA.- 3.1.3.2 Assay.- 3.2 Viral DNA, e.g. CMV, Enzyme Immunoassay (DEIA).- 3.2.1 CMV Infection Human Beings.- 3.2.2 Applications Cytomegalovirus Pathogenesis.- 3.2.3 Definition Infection.- 3.2.4 Diagnosis 3.2.5 3.2.5.1 Samples - Principal Difficulties.- 3.2.5.2 3.2.5.3 DEIA: Immunoassay.- 3.2.6 Limitation 3.3 HPLC-Analysis 3.3.1 3.3.2 3.3.2.1 Benefits HPLC 3.3.2.2 Drawbacks 3.4 Absolute Numbers Copies Sample 3.4.1 3.4.2 3.4.2.1 Generation an 3.4.2.2 Calibration Oligonucleotide.- 3.4.2.3 MRP 3.4.2.4 Acknowledgment.