LABORATORY TECHNIQUES FOR GENE EXPRESSION ANALYSIS
LABORATORIO DI TECNICHE DI ANALISI DELL'ESPRESSIONE GENICA
|Lecturer||Office hours for students|
|Anna Casabianca||Previous appointment by phone or email.|
Assigned to the Degree Course
|Date||Time||Classroom / Location|
|Date||Time||Classroom / Location|
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.
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.
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).
12. PCR method (2nd part).
13. Types of PCR.
13.1 Long PCR.
13.2 Nested PCR.
13.3 Multiplex PCR.
13.4 Cloning PCR.
14. Semi-quantitative PCR.
15. Real-time PCR.
16. Digital PCR.
17. Gene expression and transcriptome analysis.
18. Microarray technology.
19. Sanger Sequencing versus Next-Generation Sequencing.
20. Next-Generation Sequencing (NGS).
20.1 RNA sequencing (RNA-Seq) method.
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.
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
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