Differential-Display Reverse Transcription-PCR (DDRT-PCR) - Colonna-Romano, Sergio; Leone, Antonella; Maresca, Bruno; SpringerLink (Online service) (Berlin, Heidelberg : Springer Berlin Heidelberg : Imprint: Springer, 1998)
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.
Keywords: Cell biology; Biochemistry; Cell Biology; Biochemistry, general