Molecular techniques of RNA analysis

biology
biotechnology

elective courses
optional courses

Genetics and Genetic Engineering 1400-114GEN
Molecular Genetics 1400-215GEBM

The student should have basic knowledge of molecular biology, at least within the scope of the "Genetics and Genetic Engineering" course (course ID 1400-114GEN; or similar). Good, but not a prerequisite if the student has previously completed a course on molecular techniques, e.g. "Molecular Genetics" (course ID 1400-215GEBM; or similar).

Necessary practical skills that student should have: basics of laboratory work – sterile work with microbiological cultures, use of automatic pipettes, electrophoresis in agarose gels, preparation of simple enzymatic reactions (PCR).

Curiosity of the "RNA World" is welcome.

Classroom

The course is addressed to students of the Faculty of Biology and MISMaP, interested in RNA metabolism and modern molecular techniques used in the study of RNA.

Lectures:

1. "RNA world" part 1.

The RNA world concept. Nobel Prizes in the field of RNA. Catalytic RNA molecules - classes, mechanism, occurrence. SELEX. RNA world vestiges - ribosome, spliceosome, RNA viruses. Diversity of RNA classes in Eukaryotes and their metabolism. Basic mechanisms of gene expression regulation from transcription to translation. Alternative processes. RNA surveillance mechanisms. Ribonucleoprotein structures, cellular condensates.

2. "RNA world" part. 2.

3. "Reverse Transcription Techniques. Transcription techniques: TRO, ChIP, RIP and DIP. RNA polyA ends analysis".

Reverse Transcription (RT) – theory. Overview of RT-based techniques: RT-PCR including semi-quantitative, pulse RT-PCR, RACE, cRT-PCR, primer extension technique. Techniques for studying nascent transcripts: nuclear "Transcription Run-On", chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RiP). RNA Polymerase I transcription termination studies will illustrate the above techniques. Specificity of DNA binding by transcription factors: "ChIP on chip", DNA immunoprecipitation (DIP). Examination of polyA RNA ends (PASE technique, isolation of polyA+ RNA fraction).

4. "Non-coding RNA".

Non-coding RNA (ncRNA). Biogenesis and functions of small and long non-coding RNAs in Eukaryotic cells. Transcriptional and post-transcriptional silencing mechanisms dependent on ncRNAs. RNP complexes involved in silencing. The role of chromatin in the regulation of gene expression.

5. "RNA world" part. 3.

6. "Real-time PCR (quantitative; qPCR)".

The qPCR theory: methods of DNA detection, basics of primers design, hybridization probes, introduction to calculations. Applications: determination of the transcripts levels in the cell, detection of pathogen nucleic acids, detection of single-nucleotide polymorphisms (SNP). Good laboratory practice in qPCR experiments and the most common "pitfalls" waiting for experimenters.

7. "RNA in neurons".

mRNA transport in dendrites and axons, local translation in response to synaptic stimulation. Methods of mRNA visualization in living neuron in real time, in situ hybridization, biochemical methods for direct detection of transcripts undergoing local translation in neurons.

8. "RNA metabolism in physiological processes".

The role of RNA processes and ncRNAs in cell metabolism in plants and animals: hormone signaling, embryonic and generative development, circadian clock, resistance to stress and pathogens.

9. "Study of RNA metabolism enzymes on the example of the exosome".

RNA degradation pathways in eukaryotic organisms. Exo- and endonucleases. Exosome: a multifunctional and multicomponent complex. Structural and functional studies of the exosome in yeast and human cells. Mechanism of action, cooperation of exo- and endo-nucleolytic activity.

10. "RNA Structure vs. Function. In vitro RNA structure mapping. Methods of studying transcriptomes".

In vitro RNA structure mapping: molecular probes that specifically digest RNA structure and sequence. Riboswitches and aptamers – natural and selected for the needs of drugs or biosensors. High-throughput transcriptome testing methods based on next-generation sequencing techniques (RNA-seq, ChIP-seq).

11. "Methods of RNA structural biology".

Methods of studying the structures of RNA-protein complexes, methods of crystallization of RNP complexes (on the example of the ribosome, snRNP, RNase H). Comparison of crystallography and NMR studies for RNA. SAXS technique. Chemical (SHAPE) and bioinformatics prediction of RNA structures. Electron microscopy technique at low temperatures (Cryo-EM).

12. "Global analyzes of ribonucleoprotein complexes" part 1.

Biochemical methods of ribonucleoprotein (RNP) complexes purification. RNA chromatography and high-throughput RNP methods combined with NGS RNA-sequencing and mass spectrometry. Transcriptome, translatome and proteome analyses. High-throughput studies of chromatin, RNA structure and RNA-RNA, RNA-DNA and RNA-protein interactions. Methods of visualizing single RNA molecules, transcription and translation in living cells. Analysis of RNP complexes in cells by proximity labeling techniques.

12. "Global analyzes of ribonucleoprotein complexes" part 2.

Part of the lectures will be conducted by invited guests.

Practical part of the course (classes/lab work):

1. Basics of working with RNA. RNA isolation from yeast and plants.

2. RNA quality assessment. RNA electrophoresis. Radioisotope and fluorescent methods of RNA detection. miRNA detection in plants - introduction. miRNA detection in plants, Northern-blot technique (1) with RNA separation in polyacrylamide gel.

3. Northern-blot technique (1): hybridization with a biotin-labeled oligonucleotide probe. Washing, scanning and analysis of results. Detection of CUT transcripts in yeast: Northern-blot technique (2) with RNA separation in agarose gel.

4. Detection of CUT in yeast: radioactive probe labeling by "asymmetric PCR" and hybridization. Analysis of scientific images.

5. Detection of CUT in yeast: discussion of Northern-blot hybridization results (2). Analysis of the 3' ends of small nucleolar RNAs (snoRNAs) by cRT-PCR. Digestion of RNA-oligo nucleotide duplexes with RNase H and ligation (circularization) of RNA.

6. Analysis of the 3' ends of the snoRNA: RT + PCR.

7. cRT-PCR: separation of products in the gel and analysis of results. mRNA detection by RT-qPCR. Designing primers for qPCR - theory and practice. “Competition” for the most efficient pair of starters, i.e. each student will design at least 1 pair of starters, which will be ordered and tested during the next class.

8. Performing the qPCR reaction: testing the designed primers for chosen genes.

9. Analysis of qPCR results.

10. Biochemical determinations of the activity of RNA degrading enzymes. Analysis of the ribonucleolytic activities of different versions of the Dis3 protein - the main catalytic subunit of the exosome complex; testing the sensitivity of the 5'-3' exoribonucleolytic activity of the Xrn1 protein to the phosphorylation status of the 5' end of the substrate; degradation reactions of fluorescently labeled synthetic RNA oligonucleotides, separation of reaction products in a denaturing polyacrylamide gel.

11. Exploratory analysis of transcriptomic and translatomic data from RNA-seq experiments - bioinformatics exercises.

12. EMSA. Interactions of proteins and nucleic acids. Binding of yeast Nsi1 protein to rDNA. Fluorescent "gel-shift" (incubation of samples, separation in native polyacrylamide gel, scan).

13. Students’ presentations to pass the practical part of the course.

Course book and textbooks "Genomes" T. Brown, "Genetyka molekularna" ed. P. Węgleński, “Fundamental Molecular Biology” L. A. Allison; supplementary literature: scientific publications (experimental and review) indicated during the classes by the lecturers; "Molecular Cloning: A Laboratory Manual" 3rd edition by J. Sambrook and D. Russell or 4th edition by M. R. Green and J. Sambrook.

Has knowledge of RNA metabolism in Eukaryotes.

Knows the stages of expression of different classes of RNA, with particular emphasis on co- and post-transcription processes, can give examples of RNA-protein interactions and the involvement of RNA molecules in physiological processes.

Has knowledge of RNA modifying enzymes.

Has theoretical and practical knowledge that allows to interpret phenomena and processes related to RNA metabolism based on experimental results.

Independently plans and conducts basic types of experiments.

Knows vocabulary related to RNA metabolism in both Polish and English, can communicate and discuss topics related to RNA metabolism.

Knows the theoretical basis of molecular techniques used to study RNA, to the extent that allows to understand scientific publications in this field.

Is able to critically analyze the received data and present them reliably, which contributes to maintaining the professional ethos and develops the principles of professional ethics.

Learning outcomes in English:

Uses scientific and popular science biological texts in Polish and English and communicates in English at the B2+ level (K_U03 Bi2).

Has the ability to prepare and deliver oral presentations in Polish and English at the B2+ level (K_U11 Bi2).

Has the ability to use English to read scientific literature and communicate with foreigners (K_U02 Bt2).

Conditions for passing the practical part of the course (classes):

- classes can be passed only if the student participated in not less than 85% of the classes,

- classes are graded. The grade consists of points obtained for student presentations on scientific publications in which RNA techniques were used (weight in the final grade - 2/3) and points obtained from home works (weight in the final grade - 1/3),

- the topic of the final presentation is chosen by the student from the suggestions of the lecturers, or the student may propose a publication himself, but the choice must always be approved by the coordinator,

- presentations in an open style, i.e. we allow questions during the presentation from the lecturers and other participants of the course.

Conditions of the final exam:

- the subject is graded,

- only a student who has passed the classes may take the exam,

- test exam: multiple-choice test (4 variants of answers to each question),

- in order to pass the exam, the student must obtain at least 50% of the points.

Not required.