LABORATORY FOR ANALYSIS OF GENE ESPRESSION
LABORATORIO DI ANALISI DELL'ESPRESSIONE GENICA
| A.Y. | Credits |
|---|---|
| 2023/2024 | 8 |
| Lecturer | Office hours for students | |
|---|---|---|
| Anna Casabianca | Previous appointment by phone or email. |
| Teaching in foreign languages |
|---|
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Course with optional materials in a foreign language
English
This course is entirely taught in Italian. Study materials can be provided in the foreign language and the final exam can be taken in the foreign language. |
Assigned to the Degree Course
| Date | Time | Classroom / Location |
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| Date | Time | Classroom / Location |
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Learning Objectives
The course aims to provide students theoretical knowledge and practical skills of techniques for the analysis of gene expression. The course will provide an overview of classical techniques (Hybridization Method, Northern Blotting) for the detection / measurement of mRNAs and the most modern techniques (real time qPCR) of accurate quantification of single gene expression levels. Notions on microarray and RNA-Seq techniques (based on NGS sequencing) will be provided for the evaluation of different profiles of gene expression (transcriptome). The laboratory experience will concern the organization of a strategy to evaluate the expression levels of potential target genes involved in a physiological or pathological process. To obtain results biologically significant of gene expression, housekeeping genes to normalize the data and appropriate statistical tests will be used.
Program
Lectures (to support laboratory activities)
1. laboratory organization for a correct sperimentation in molecular biology and biochemistry.
1.1 Organization of laboratory areas with a unidirectional correct workflow.
1.2 Standard Operating Procedure.
1.3 Workflow.
2. Preparation and storage of biological samples for nucleic acid analysis.
3. Calculations used in molecular biology laboratories (biochemistry).
4. Sistemi di quantificazione degli acidi nucleici.
4.1 Metodo spettrofotometrico. NanoDrop 1000.
4.2 Metodo colorimetrico.
4.3. Metodo fluorimetrico.
5. RNA Extraction.
5.1 RNA manipulation / precautions. Prevention of endogenous RNase contamination.
5.2 Appropriate choice of method (classic or commercial kit).
5.3 Main steps of RNA isolation from various matrices.
6. Qualitative analysis of RNA.
6.1 Agarose denaturing gel electrophoresis.
6.2 Bioanalyzer RNA.
7. mRNA purification.
7.1 Affinity chromatography for the separation of poly (A) + RNAs.
8. cDNA synthesis strategies.
9. Blot Transfer of Nucleic Acids to a Membrane
9.1 Northern Blot technique.
9.2 Probe labelling.
9.3 Hybridization procedure.
9.4 Dot blot.
10. Reverse Transcriptase PCR.
10.1 One step PCR.
10.2 Two step PCR.
11. PCR method (1st part).
11.1 Characteristics.
11.2 Theory.
12. PCR method (2nd part).
12.1 Optimization.
13. Types of PCR.
13.1 Long PCR.
13.2 Nested PCR.
13.3 Multiplex PCR.
13.4 Cloning PCR.
14. Semi-quantitative PCR.
14.1 Characteristic.
14.2 Examples.
15. Real-time PCR.
Laboratory activities
1. Objective of gene expression analysis and work strategy design.
2. Selection of housekeeping gene for data normalization.
3. Expression profile evaluation of target genes.
5. Isolation of total RNA from the selected matrix.
6. Spectrophotometric analysis.
7. Synthesis of cDNA by reverse transcription (RT).
8. Dilutions, preparation of qPCR reactions.
9. Data analysis of results.
10. Final report/presentation.
Bridging Courses
A good knowledge of basic Molecular Biology is required to understand the course contents.
Learning Achievements (Dublin Descriptors)
D1 - Knowledge and ability of comprehension. Students will have to demonstrate that they have acquired the theoretical-practical knowledge of the experimental techniques and be able to organize the experimental design for an analysis gene expression study. Finally, students will have to be able to develop an appropriate analysis of the results to understand their biological significance.
D2 - Ability to apply knowledge and comprehension. Students will have to demonstrate that they have mastered the methodologies covered during the course and to evaluate their use in an experimental context, to develop a correct experimental design related to gene expression analysis and to evaluate their biological significance.
D3 - Autonomy of judgement. Students must be able to evaluate and apply the methodological knowledge obtained during the course and be able to independently process the results obtained in the experimental phase.
D4 - Communication skills. Students will have to demonstrate that they have the ability to independently find the scientific publications related to the course, and the ability to improve their theoretical and practical knowledge in the field of gene expression analysis technologies.
D5 - Learning skills. Students will demonstrate the ability to independently increase the basic knowledge of newly emerging fields of molecular biology
Teaching Material
The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it
Teaching, Attendance, Course Books and Assessment
- Teaching
Learning activity will mainly consist of lab experiences. Lectures will be intended to supply the theoretical knowledge of the leading techniques employed in analysis of gene expression.
The schedule will be introduced at the beginning of the course.
- Attendance
It is strongly suggested that students attend the lab hours.
- Course books
R. J. Reece. ANALISI DEI GENI E DEI GENOMI, EDISES, 2011.
T. A. Brown. BIOTECNOLOGIE MOLECOLARI, Principi e tecniche, Zanichelli, 2017.
F. Amaldi, P. Benedetti, G. Pesole, P. Plevani. tecniche e metodi per la Biologia molecolare, CEA casa editrice ambrosiana
- Assessment
The final assessment consists of a written multiple choice test and oral exam. Students will elaborate an oral presentation on their laboratory experiments in appropriate scientific language.
- Disability and Specific Learning Disorders (SLD)
Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.
To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.
Additional Information for Non-Attending Students
- Teaching
Learning activity will mainly consist of lab experiences. Lectures will be intended to supply the theoretical knowledge of the leading techniques employed in analysis of gene expression.
The schedule will be introduced at the beginning of the course.
- Attendance
It is strongly suggested that students attend the lab hours.
- Course books
R. J. Reece. ANALISI DEI GENI E DEI GENOMI, EDISES, 2011.
T. A. Brown. BIOTECNOLOGIE MOLECOLARI, Principi e tecniche, Zanichelli, 2017.
F. Amaldi, P. Benedetti, G. Pesole, P. Plevani. tecniche e metodi per la Biologia molecolare, CEA casa editrice ambrosiana
- Assessment
The final assessment consists of a written multiple choice test and oral exam. Students will elaborate an oral presentation on their laboratory experiments in appropriate scientific language.
- Disability and Specific Learning Disorders (SLD)
Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.
To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.
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