University of Siena (UNISI)

http://www.dbm.unisi.it

annalisa.ciabattini@unisi.it, santorof@unisi.it, Francesco.iannelli@unisi.it

The University of Siena, one of the oldest and first publicly funded universities in Italy founded in 1240, has about 2100 employees and a turnover of €400 million. The Department of Medical Biotechnology consists of many research groups covering biotechnology, vaccine development, infectious disease, immunopathology, microbiology, genetics and microbial immunity. The Laboratory of Molecular Microbiology and Biotechnology is part of this Department, and its research focuses on microbial genetics, host-pathogen interaction, recombinant vaccines and vaccine immunology.

Role in the TRANSVAC project

UNISI is WP Leader of WP13 TNA7 “Immunocorrelates and Systems Biology”; and partner of WP5 JRA4 “Immuno-correlates and Systems Biology”, WP3 JRA1: Improved optimisation and harmonisation of cross-vector screening, WP7, TNA 1 “Cross-platform screening and optimisation service” and WP6 “JRA5: Animal models”

Main staff involved in TRANSVAC

Gianni Pozzi, Prof.; Donata Medaglini, Prof.; Francesco Iannelli, Prof.; Annalisa Ciabattini, PhD; Elena Pettini, PhD; Fabio Fiorino, PhD; Francesco Santoro, MD, PhD; Alessia Donato, PhD student; Sara Sorgi, PhD student; Simone Lucchesi, PhD student; Simone Furini; Marco Valentini; Lorenzo Bruttini; Alberto Balistreri; Antonio Vicino.

The Facility

UNISI has established a NGS platform based on Ion Proton instrument (Thermo Fisher) for targeted RNA sequencing. Laminar flow cabinets are present in a BLS2 area for RNA extraction from blood. For RNA quality and quantity assessment we have a Qubit fluorimeter and an Agilent Bioanalyzer. Two advanced technologies for detecting and measuring multiple soluble biomarkers in soluble samples are also available in our facility, the Luminex xMap and flow cytometric CBA assay. Both systems can simultaneously detect many targets such as cytokines, chemokines and inflammatory biomarkers in a single sample that can be serum, plasma and tissue culture supernatants. Multiplexing analysis is especially useful when only a small amount of sample is available, maximizing the number of proteins that can be analysed. These technologies are particularly powerful when looking for changes in concentrations of multiple targets under specific conditions, such as following a vaccine administration, infection, or a drug treatment. The assays reduce sample requirements and time to results in comparison with traditional ELISA and Western blot techniques. The laboratory has also all of the technologies required to produce recombinant bacteria expressing vaccine antigens to be used as non-pathogenic vaccine vectors. The laboratory has all of the technologies required to perform molecular work including gradient polymerase chain reaction (PCR) blocks, RT-PCR and FACS. UNISI hosts a unit of Systems Biology and Bioinformatics, equipped with the necessary infrastructure for data storage and high-performance computational analyses. The main areas of activity of the unit are the analysis of NGS experiments, and the mathematical modelling of biological systems at the cellular and molecular level.

TNA or Training provided

UNISI provides expertise in the determination of transcriptomic profiles of blood samples using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit to prepare NGS libraries to be sequenced on the Ion Proton™ System. The expression level of 20,802 human genes is quantified by this approach, using as an input 10-100 ng of total RNA. The minimum guaranteed output is 8 million reads per sample. UNISI provides expertise in the measurement of cell signalling proteins, such as cytokines, chemokines and inflammatory biomarkers in multiple samples (including serum, plasma and tissue culture supernatants), using a multiplex suspension array system or a flow cytometric bead assay. Both technologies allow the simultaneous detection of multiple factors in serum, plasma and tissue culture supernatants. Moreover, UNISI produces recombinant bacteria expressing vaccine antigens to be used as non-pathogenic vaccine vectors. Services include the design and construction of new recombinant bacterial strains expressing requested proteins (i.e. vaccine antigens) and assessment of the surface expression of the protein by flow cytometric analysis. The unit of Systems Biology and Bioinformatics provides expertise in the analysis of RNA sequencing experiments. Taking as inputs raw-reads from NGS experiments, the unit can perform the following analyses: 1) identification of statistically relevant differences in expression levels; 2) dimensionality reduction; 3) supervised and unsupervised clustering of expression data.

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