Differential-Display Reverse Transcription-PCR (DDRT-PCR)

Overview.- 1 Differential Display of mRNAs: Principles and General Applications.- 2 DDRT-PCR: A Brief Description.- 3 Summary.- 1 Preparation of Total RNA.- 1.1 Extraction from Yeast by Phenol.- 1.2 Extraction by Guanidine Thiocyanate, Plant Tissue.- 1.3 Extraction by Guanidine Thiocyanate, Animal Cells.- 1.4 Extraction from Fish Liver.- 1.5 RNA Quantification.- 1.6 DNase Treatment.- 1.7 Verification of Integrity.- 2 Differential Display.- 2.1 Reverse Transcription of Total RNA.- 2.2 PCR Amplification.- 3 Size Separation of cDNA Fragments.- 3.1 Gel Electrophoresis.- 3.2 End Labeling of DNA Markers.- 4 Isolation of Differentially Expressed cDNA Fragments.- 4.1 Alignment of a Differential-Display Gel Using a Film Image.- 4.2 Elution of cDNA Fragments from Gel.- 5 Reamplification of Eluted cDNA.- 6 Cloning of Amplified cDNA Fragments.- 6.1 Purification of cDNA Fragments from Agarose.- 6.2 Ligation of cDNA into Vectors.- 6.3 Preparation of CaCl2 Competent Cells for Transformation.- 6.4 Preparation of Electrocompetent Cells.- 6.5 Transformation of CaCl2 Competent Cells.- 6.6 Transformation of Competent Cells by Electroporation.- 7 Checking Subcloned Fragments.- 8 Confirmation of Differential Expression of Cloned cDNA Fragments.- 8.1 Slot and Northern Blots.- 8.2 DNA Labeling and Hybridization.- 8.3 Ribonuclease Protection Assay.- 8.4 Nuclear Run-On Assay.- 9 Northern Blot Affinity Capturing of cDNA.- 9.1 PCR Labeling of Differentially Expressed cDNA.- 9.2 Northern Blot Affinity Capturing.- 10 Sequencing of Differentially Expressed cDNA Fragments.- 10.1 Plasmid DNA Preparation.- 10.2 Sequencing Reaction with Sequenase Version 2.0 T7 DNA Polymerase.- 10.3 Sequencing Reaction with Thermo-Sequenase Cycle-Sequencing Kit.- Materials, Equipment, Reagents, and Suppliers.- References.