PřF:Bi5599 App. Biochem. and Cell Methods - Course Information

Bi5599 Applied Biochemistry and Cell Biology Methods

Extent and Intensity

2/0/0. 2 credit(s) (plus extra credits for completion). Type of Completion: zk (examination).
In-person direct teaching

Teacher(s)

doc. RNDr. Alena Hyršlová Vaculová, Ph.D. (lecturer)
Mgr. Jiřina Medalová, Ph.D. (lecturer)
RNDr. Vendula Hlaváčková Pospíchalová, Ph.D. (lecturer)
Mgr. Pavel Kadeřávek, Ph.D. (lecturer)
doc. Mgr. Karel Souček, Ph.D. (lecturer)

Guaranteed by

doc. RNDr. Alena Hyršlová Vaculová, Ph.D.
Department of Experimental Biology – Biology Section – Faculty of Science
Contact Person: Mgr. Jiřina Medalová, Ph.D.
Supplier department: Department of Experimental Biology – Biology Section – Faculty of Science

Timetable

Thu 11:00–12:50 B11/335

Prerequisites (in Czech)

Bi4020 Molecular biology && C3580 Biochemistry

Course Enrolment Limitations

The course is also offered to the students of the fields other than those the course is directly associated with.

fields of study / plans the course is directly associated with

there are 6 fields of study the course is directly associated with, display

Course objectives

The main goal of the course is to enable students to understand the importance of individual chemical/biochemical techniques and to interpret both their own experimental data, based on the techniques taught in the course. They should be able to work with the information in the primary literature, in order to understand and implement the techniques described in primary scientific literature in the area of animal physiology and molecular physiology. Based on this, they will be able to present and explain the techniques used in their own work to expert audience.

Learning outcomes

At the end of the course, students will be able to: identify and describe basic methodology for determination of protein levels and localization, estimation of protein-protein interactions, functional analysis of protein domains, interactions of proteins with DNA; describe basic principles of manipulation of protein expression and function; describe principles of isolation of nucleic acids and determination of gene expression; describe methodology for analyses of lipids and small organic molecules based on chromatography and mass spectrometry; describe basic chemical and biochemical methods used in primary literature.

Syllabus

1. Fundamentals of Working with Cell Cultures, Processing of Cellular Material. Growth of cell cultures, culture media, types of cultivation, passaging and preservation of cell lines, quality control, contamination prevention, evaluation of basic cytokinetic parameters (proliferation, viability). 2. Selected Protein Separation Methods, Electrophoretic Techniques. Electrophoresis – basic types and principles. SDS-PAGE, Western blotting, options for immunodetection, antibodies. Isoelectric focusing (IEF). 2D-electrophoresis. 3. RNA – Isolation, Detection (qRT-PCR, RNA-seq, Microarrays), Non-Coding RNA, si/shRNA. RNA world hypothesis, RNA functions, RNA isolation, use of mRNA for qRT-PCR, next-generation sequencing, RNA sequencing, microarrays, manipulation of gene expression via RNA – antisense oligonucleotides, RNA interference, functions of non-coding RNAs, clinical use of miRNA, use of si/shRNA in research. 4. Analysis of Protein Modifications, Protein Complexes, Protein Expression Manipulation, and Proteomics. Post-translational modifications (PTMs) of proteins, detection of modifications in endogenous proteins (e.g., phospho-specific antibodies), (Chromatin) Immunoprecipitation, affinity purification, experimental cellular systems for studying protein function (GOF – constitutive vs. inducible expression, LOF – CRISPR/Cas9), analysis of complex protein mixtures (BioID, mass spectrometry), application of these techniques to biological questions. 5. Lipids – Selected Separation Techniques and Detection Principles (GC, HPLC, and MS) Overview of separation techniques combined with mass spectrometry used in the analysis of biological samples. A brief summary of ionization principles (ESI, APCI, APPI, ICR, MALDI-TOF) and their application in so-called omics techniques. Practical demonstration of GC/MS, LC/MS-MS, and LC/Hybrid MS techniques. Examples of the use of current separation techniques and detection principles. 6. Preparation, Characterization, and Testing of Nanoparticle-Based Drug and Vaccine Carriers 7. Fluorescence Techniques – Flow Cytometry, Antibodies and Their Use in Cell Biology. Flow cytometry methods, their principles and applications in cell analysis. Techniques for measuring size, morphology, cell cycle phase, DNA content, and presence of specific proteins on the cell surface or in the cytoplasm. Role of flow cytometry in medical diagnostics, cell biology research, and analysis of heterogeneous cell populations, including sorting of live cells for further analysis. 8. Lipidomics – Chromatographic Methods, Study of Lipid Expression and Localization in the Cell. Methods for lipid analysis such as HPLC, mass spectrometry, and chromatography. Techniques for lipid extraction from cells and tissues (Bligh & Dyer method), methods for detecting different lipid types. Lipid functions in cells, in cellular membranes, energy metabolism, and signaling processes. 9. DNA – Structure and Replication, DNA Isolation, DNA Separation (Gel Electrophoresis, Capillary Electrophoresis, Advanced Electrophoresis), PCR (Standard, Quantitative, Specialized Variants), LAMP, DNA Fragment Analysis (Hybridization, RFLP, FISH), DNA Sequencing, Manipulation of Gene Expression, Genetic Engineering, Gene Knockout (Homologous Recombination, Cre-LoxP System, FLP/FRT System, CRISPR/Cas9 System), Gene Overexpression – Plasmids and Transfection. 10. NMR Theory and Excursion. Principle of NMR (nuclear spin, macroscopic magnetic moment), description of basic NMR experiments, nuclear spin magnetic moment interactions and their implications, NMR spectra of biomolecules, introduction to multidimensional NMR spectroscopy, principles of NMR frequency assignment, basic structural information from NMR (NOE), principles of calculating biomolecular structures using NMR, studying interactions via NMR, NMR and biomolecular dynamics. Excursion: demonstration of NMR spectrometers and related equipment, basic technical description of NMR spectrometers and probes. 11. Application of In Vivo Mammalian Models in Physiology. Ethical and legislative aspects of working with animals (3+1 R principles), use and selection of appropriate animal models, common mammalian models in physiology (biomedicine), laboratory rat, laboratory mouse, housing of laboratory mice (conventional, SPF, gnotobiotic), experiments with laboratory mice, genetic manipulation of laboratory mice – commonly used methods and resources (mouse strains) – natural and induced mutations, gene targeting in embryonic stem cells, "knock-out and knock-in" mice, Cre/loxP system, new gene-targeting methods – ZFN, TALEN, and CRISPR/Cas9, transgenic mice, mouse clinics. 12. Omics Technologies – Genomics, Transcriptomics, Metabolomics, Databases, Personalized Medicine, and Big Data. Overview of omics technologies + big data – information pyramid, data sharing and use – biomarkers and personalized medicine. Genomics: Sanger vs. NGS sequencing – whole genome vs. exome vs. SNP genotyping, COSMIC and TCGA databases. Transcriptomics: Microarrays vs. RNA sequencing, gene set analysis and pathway analysis, Reactome. Metabolomics, single-cell omics and multi-omics (e.g., scRNA-seq).

Literature

• Shapiro HM. Practical Flow Cytometry, 4th Edition, Wiley 2003.
• SIVAKAMAVALLI, Jeyachandran a Baskaralingam VASEEHARAN. An overview of omics approaches: Concept, methods and perspectives [online]. In: Recent trends in biochemistry. Yeosu (South Korea): MedDocs Publishers LLC, 15. 6. 2020.
• Wilson et al.: Principles and Techniques of Practical Biochemistry, 5th ed., CUP, 2000
• Shuken, S. R. An Introduction to Mass Spectrometry-Based Proteomics. Journal of Proteome Research, 2023, 22(6), 2151–2171. DOI: 10.1021/acs.jproteome.2c00838. [online] Dostupné z: https://pubs.acs.org/doi/10.1021/acs.jproteome.2c00838.
• E.K. Neumann: Spatial Multiomics Toward Understanding Neurological Systems. Journal of Mass Spectrometry, 2025; 60:e5143. Wiley.
• EVROPSKÁ UNIE. Směrnice Evropského parlamentu a Rady 2010/63/EU ze dne 22. září 2010 o ochraně zvířat používaných pro vědecké účely [online]. In: Úřední věstník Evropské unie. 20. října 2010, L 276, s. 33–79.
• SIVAKAMAVALLI, Jeyachandran, Kiyun PARK a Ihn-Sil KWAK. Genome databases, types and applications: An overview [online]. In: Recent trends in biochemistry. Yeosu (South Korea): MedDocs Publishers LLC, 7. července 2020.
• Ďurech, M., Trčka, F., Vojtěšek, B., Müller, P. Metody pro studium protein-proteinových a protein-ligandových interakcí. Klinická onkologie, 2014, 27(Suppl 1), S75–S81. [online]. Dostupné z: https://www.linkos.cz/files/klinicka-onkologie/395/4488.pdf
• Alberts et al.: Molecular Biology of the Cell, 4th ed., Garland Science, 2002
• Bonifacino JS. Short Protocols in Cell Biology, Wiley 2004.
• Hedrich, H. The Laboratory Mouse. 3. vyd. London: Academic Press, 2012.
• M Duttb et al.: Chemical Ionization Mass Spectrometry: Fundamental Principles, Diverse Applications, and the Latest Technological Frontiers. Mass Spectrometry Reviews, 2025; 1–22. Wiley.
• C. Dass: Fundamentals Of Contemporary Mass Spectrometry, 2007, Wiley.
• Klouda, Moderní analytické metody, Pavel Klouda, Ostrava, 2003
• SINGH, Komal P.; Christine MIASKOWSKI; Anand A. DHRUVA; Elena FLOWERS and Kord M. KOBER. Mechanisms and Measurement of Changes in Gene Expression. BIOLOGICAL RESEARCH FOR NURSING. THOUSAND OAKS: SAGE PUBLICATIONS INC, 2018, vol. 20, No 4, p. 369-382. ISSN 1099-8004. Available from: https://doi.org/10.1177/1099800418772161. info
• GAJ, Thomas; Shannon J. SIRK; Sai-lan SHUI and Jia LIU. Genome-Editing Technologies: Principles and Applications. COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY. COLD SPRING HARBOR: COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2016, vol. 8, No 12, 20 pp. ISSN 1943-0264. Available from: https://doi.org/10.1101/cshperspect.a023754. info

Teaching methods

According to the current measures of Masaryk university the course will be taught distantly via MS Teams - either by on-line lectures synchronized with time schedule, or by commented presentations followed by on-line consultations. Students will be informed in case of any change in advance.

Assessment methods

Course is finished by written test going through the methods demonstrated, at least 60% of correct answers is necessary to pass. In case of distant learning the testing will be performed by IS questionaries.

Language of instruction

Czech

Further comments (probably available only in Czech)

Information on course enrolment limitations: Na předmět se vztahuje povinnost registrace, bez registrace může být znemožněn zápis předmětu!

Listed among pre-requisites of other courses

• Bi5599c Applied Biochemistry and Cell Biology Methods - practice
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