The course is also offered to the students of the fields other than those the course is directly associated with.
The capacity limit for the course is 24 student(s).
Current registration and enrolment status: enrolled: 18/24, only registered: 0/24, only registered with preference (fields directly associated with the programme): 0/24
Fields of study the course is directly associated with
The objective is to introduce students into the methodological principles of molecular medicine with special view on the use of molecular biological methods in cancer diagnosis and therapy individualization. Students will be introduced into design of the study, experimental techniques, statistical analysis, and implementation to the clinical practice.
1. Introduction to the basic methodological approaches in molecular medicine, general principles of work in the molecular biological laboratory, demonstration of instruments, inspection of laboratories and facilities used in molecular medical research.
2. Introduction to the principles of study design in molecular medicine, examples of model studies, identification of diagnostic, prognostic, and predictive biomarkers, estimation of sample size required to achieve statistical significance, selection of suitable experimental approaches, availability of biological material, high-throughput analysis=screening and validation, control systems, time schedule, budget calculation. Detailed study design according to the lecturer instruction.
3. Types of biological material, the basic principles of work with biological material in molecular medicine (storage, tissue homogenization, cell separation, selection of the optimal procedure for the isolation of nucleic acids). Tissue homogenization, isolation of nucleic acids (DNA, RNA) and purity (ThermoScientific NanoDrop) and integrity control (LOC technology - Agilent Bioanalyzer, gel electrophoresis).
4. High-throughput analyses - advantages and disadvantages of individual approaches, a global analysis of gene expression (Affymetrix platform) and microRNA expression profiles (qRT-PCR arrays - TaqMan Low-Density Arrays), introduction to the evaluation of experimental data, identification of potential biomarkers, real experimental data analysis.
5. Validation I - the use of individual qRT-PCR assays on large independent sets of patients in accordance with the design of the study. Examples of experimental design Real-Time PCR, multiple-level control systems. Reverse transcription.
6. Validation II - the use of individual qRT-PCR assays on large independent sets of patients in accordance with the design of the study. Real-Time PCR.
7. Statistical analysis of data obtained from the validation. Introduction to the biostatistic methods (Mann-Whitney test, Wilcoxon test, multivariate analysis (Cox regression model), the ROC analysis - sensitivity, specificity, Kaplan-Meier survival analysis, ...).
8. Functional validation of biomarkers. In vitro experiments, general principles of work in the cell culture laboratory, and design of experiments with cell models.
9. Cell cultures cultivation, cell growth curves analyses and cytotoxicity tests (IC50).
10. Cell transfection with synthetic oligonucleotides to suppress selected genes and miRNAs on transcription levels. Evaluation of transfection efficiency (qRT-PCR, western blot).
11. Effect of transfection on cell viability, apoptosis and cell cycle (MTT assay, flow cytometry - Annexin V and propidium iodide).
12. Overall assessment of the study, final protocol and recommendations for possible implementation of tested biomarker into clinical practice.
ZIMA, Tomáš. Laboratorní diagnostika. 2., dopl. a přeprac. vyd. Praha: Galén, 2007. xxxviii, 9. ISBN 9788024614236. info
Twelve exercises have to be completed according to the schedule.
Credit completion on the base of participation and protocol processing.