Portale di Ateneo - En.Unibs.it

LABORATORY OF CLINICAL PATHOLOGY

General Module

Course Sheet

Academic Year of enrolment:

2012/2013

Academic Year of provision:

2014/2015

Type of Course:

Characterising

Degree:

Bachelor's Degree Programme in BIOTECHNOLOGIES

Disciplinary Sector:

Clinical Pathology

Is part of:

CLINICAL PATHOLOGY AND ANIMAL MODELS

Grouping type:

Credits:

Programme Year:

Professor and Collaborators:

FRA Annamaria Professor

Cycle:

First semester

Hours of classroom activity:

Hours of individual study:

Educational Goals

The course aims to provide students with the theoretical and practical bases of some techniques used in a biomedical laboratory. Although the course is meant to be mainly practical, the students are encouraged to deepen the theory behind the experimental protocols, their critical aspects and possible alternative approaches.

Content

The course consists of practical laboratory work on some techniques used in biomedical research. The techniques will be applied to the analysis of secretion of pathological variants of alpha1-antitrypsin, expressed in a cell model.
In detail we will perform:
A) transfection of eukaryotic cells. A murine hepatoma cell line (Hepa1.6) will be transfected with expression vectors encoding for the wild-type or mutant alpha1-antitrypsin variants. The transfection efficiency will be assessed transfecting in parallel a vector encoding beta-galactosidase.
B) RNA extraction. Total RNA will be extracted from transfected cells, quantified by NanoDrop spectrophotometer and analyzed by agarose gel electrophoresis.
C) RT-PCR. The levels of mRNA expression of the different alpha-antitrypsin variants will be quantified by RT-PCR. The RNA extracted from transfected Hepa1.6 cells will be reverse transcribed and the mRNA of alpha1-antitrypsin and actin will by analysed by PCR with specific primers.
D) ELISA. The efficiency of secretion of the different variants of alpha1-antitrypsin will be evaluated by analyzing their levels in the supernatants of transfected cells by sandwich ELISA.

Recommended Bibliography

A booklet prepared by the teacher on the experiments’ protocols. Lecture notes. Methodological material on specific techniques.

Teaching Methods

The course consists of one teaching block of 30 hours divided into five days and takes place in the teaching laboratories of DMMT. The students will perform transfections of eukaryotic cells, RNA extractions and RT-PCR, protein quantification by ELISA. These techniques are organised in a series and simulate a real complex "experiment", including the final analysis and presentation of results. The course includes also a few lessons dealing with theoretical aspects of the techniques used in the laboratory.

Assessment Methods

The student will have to prepare a brief report of the experiments carried out and the results obtained. The final exam will focus on the theoretical aspects of a technique among those applied during the course.

Assessment:

Final Mark

Programme of Educational Activities

Start date of teaching period:

Monday, October 6, 2014

End date of teaching period:

Friday, January 30, 2015