The Cell Culture and Genotyping Platform (CCGen) aims to provide i3S researchers state-of-the-art advances in cell culture, genotyping and gene expression technology. The facility offers expert consultation, cell culture training, genotyping and gene expression.

Tailor-made conditions are available for culture of organoids, cell lines, primary cells, clinical samples and virus, including lentivirus-based transfection service. Training is mandatory to access this facility.

Mouse genotyping is offered to all researchers who need technical expertise including protocol implementation and optimization. These consist in DNA extraction, DNA fragment amplification, detection, analysis and interpretation of results. Researchers may access our on-line ordering through the Animal Facility to request the mouse genotyping service. Urgent genotyping may also be requested by all investigators in need of a specific time point or to select primary cultures.

CFX96 and CFX384 qPCR equipments are available with thermal validation and quality control. We also support analysis of the RNA integrity by Experion automated electrophoresis system for gene expression studies.

Highly qualified experts are available for consulting all the scientific community and help solving questions that may arise concerning technical and/or data interpretation.

Gene Expression

The qPCR was developed to rapidly and accurately determine the relative amount of RNA or DNA in a sample. To accomplish rapid quantitation and efficient PCR conditions, reactions were design to produce small PCR products (often under 80-150 bp) using short cycles (usually under 30 seconds for a complete cycle). Data is generated as the reaction is running (threshold cycles, CT) or immediately after cycling (melt curves and standard curves generated from CT values). No post-reaction gel is required. Real-time PCR or quantitative PCR (qPCR) is routinely used in laboratories to help identify pathogens, genotype infectious agents, and for cancer diagnostics.

The Experion is an automated system for nucleic acid and protein analysis, which incorporates LabChip technology into an integrated system that performs multiple electrophoresis steps in one. This automated gel electrophoresis system is used for analysis (quantity and quality) of RNA, DNA and protein. Reduces the exposure to hazardous formaldehyde and ethidium bromide, decreases the amount of sample needed and provides a reliable system for documentation of results. This instrument serves as a complement to the qPCR, allowing for the fast and economic assessment of RNA quality.

Genotyping

The service provides the implementation, optimization and manual execution of animal (mouse, zebrafish…) genotyping protocols, including DNA extraction, DNA fragment amplification, detection, analysis and interpretation of the results.

Cell Culture

The facility offers well established and tailor-made conditions for culture of organoids, cell lines, primary cells, clinical samples and virus. Consultationand training are given when required. Training is mandatory for all new users (e-lesson). 
The cell culture provides Zoe Fluorescent Cell imager, an inverted imaging system with light field and three fluorescent channels, suitable for image cell culture in routine applications.

Gene Expression

• CFX96 Real time System (Bio-Rad) in operation since 2014;
• CFX384 Real time System (Bio-Rad) in operation since 2019;
• Experion, Automated Electrophoresis System (Bio-Rad) in operation since 2010.

Genotyping

• T100 thermocycler (Bio-Rad) in operation since 2011;
• 2 TGradient UNO96 (VWR) in operation since 2019;
• Termomixer compacto (Eppendorf) in operation since 2008;
• Termomixer compacto (Eppendorf) in operation since 2011;
• Dry Bath FB15103 (Fisher Scientific, Lda) in operation since 2014;
• Centrifugue MicroStar 17R (VWR) in operation since 2019;
• Centrifugue MULTIFUGE X1R 230V RESEARCH (Thermo Scientific) in operation since 2011;
• Gel Tray, 15x20 cm and 10x15 cm (BioRad) in operation since 2008;
• GelDoc EZ System (BioRad) in operation since 2012;
• Vortex Heidolph in operation since 2007.
• Vortex Mixer (Labnet) in operation since 2007.

Cell Culture

• Six CO2, heated incubators (Heraeus, Hera Cell), distributed by five independent labs in operation since 2004;
• Five laminar flow hoods (Heraeus, Hera Safe; Kojair, GL130; Thermo Scientific, MSC Advantage 1.2), one per each labs in operation since 2004;
• An inverted microscope (Zeiss, Axiovert 25) in operation since 2003;
• One heated water bath (DIG 26L AMB +5 TO 99C. SA, Grant) in operation since 2016;
• One refrigerated centrifuge (Heraeus, Megafuge 1.0R) in operation since 2004;
• ZOE Fluorescent Cell Imager (BioRad) in operation since 2015;
• Minispin plus (Eppendorf) in operation since 2007.

Cell Culture Facility

• Organoids
• Cell Lines
• Primary Cells
• Clinical Samples
• Virus

Mycoplasma detection

Real Time PCR or quantification PCR (qPCR)

Genotyping

• DNA extraction
• DNA fragment amplification
• Detection
• Analysis