Quantitation of mRNA by Polymerase Chain Reaction

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