Molecular biology

Experiments performed by the laboratory

To observe : Quantify gene and protein expression.

• RT/PCR analysis (RNA expression by semi-quantitative and quantitative methods, sequence specific amplification, sensitive detection, optimization of methodology
• WB analysis: semi-quantitative methods, optimization of methodology

To modify : Modify gene and protein expression.

• cDNA isolation
• Mutagenesis
• Constitutive or inducible expression
• Cellular cloning (lipofectant transfection of siRNA and shRNA)

To characterize : Use functional assays to observe molecular action.

• Molecular assays : immunoprecipitation, gel shifts and Dot Blot
• Cellular assays : GENE reporter

Aim of these experiments in drug development

To observe, to modify and to characterize gene and protein expression.

Gene expression and target validation = to observe

• Evaluation of expression in a panel of cultured cell lines and grafted or primary tumors
• Evaluation of gene or protein expression after in vitro or in vivo treatment
• Detection of organ invasion by tumor cells
• Evaluation of antibody specificity
• Biomarker follow-up

Structure-function studies = to modify

• Transient or stable over-expression of wild-type or mutated proteins in constitutive or inducible systems
• Decrease of expression by RNA interference: RNAse III siRNA, synthetic siRNA and plasmidic shRNA(constitutive or inducible)

Interaction studies = to characterize

• Immunoprecipitation (Tag or specific antibodies)
• Gel-shift, filter binding assay, Dot Blot, GENE reporter