RTC/RCF basic methodologies course descriptions

Course one: Genes to Proteins
Instructor: Pamela Plant

In this laboratory you will learn the basics (both theory and practice) of cloning and subcloning, gene expression in mammalian cells and Western blotting. For subcloning/cloning, you will use restriction enzyme (RE) digestion together with agarose gel electrophoresis to isolate fragments of DNA. These fragments (one from a gene of interest, the other a bacterial plasmid) will be ligated and transformed into bacteria for propagation. For gene expression, you will express this “newly formed gene” in mammalian cells using 3 different transfection methods; CaCl2, lipidbased and electroporation and will understand the difference between them and their specific applications. Finally, you will run cell lysate on a SDSPAGE and perform Western blot analysis of the resolved proteins and do a quantitation of a target band, normalized to total protein. You will learn the theory behind SDSPAGE separation of proteins and learn about numerous applications of PAGE to interrogate various biological scenarios.

Course two: Immunofluorescence in Flow Cytometry and Microscopy
Instructors: Caterina Di Ciano

In this laboratory you will learn the basics of adherent and nonadherent cell culture techniques, immunofluorescent staining (direct and indirect procedures), appropriate controls and experimental design, sample fixation, permeabilization, staining for extracellular and intracellular protein analysis, discussion of functional fluorochrome probes such as Ca2+ , pH, redox state, etc., and analysis by fluorescence microscopy and quantitative flow cytometry. For tissue culture, you will use adherent COS cells, nonadherent Jurkat (Tcell lymphoma) cells, culture and perform immunofluorescent stains for cytoskeletal structures, nuclear structures, and cell surface markers using conjugated antibodies, indirect immunofluorescence and functional fluorochromes. For microscopy, you will perform multicolour fluorescence microscopy, analyze subcellular colocalization, and understand the methodologies and hardware best utilized to address different types of biomedical hypotheses. In flow cytometry you will perform multicolour quantitative flow cytometry, compensation of overlapping fluorescent signals, dead cell exclusion, polychromatic assay design and immunophenotyping of single cells. You will further learn how to optimize, troubleshoot, analyze and interpret these experiments accordingly. Special focus will be made to the strengths and limitations of the methodologies (flow cytometry and microscopy), and when either technology (or both) is most effectively suited to your scientific project.

Course three: Bioinformatics
Instructors: Pamela Plant, Caterina DiCiano

The ability to manipulate organisms genetically has been revolutionized in the last ten years. Biological information produced by this revolution is represented in many forms: sequence data, structural data, and functional data. These data can often be accessed and interpreted only with the help of computers. Data mining is now an established tool for predicting structure and understanding function in genomics and proteomics. It is becoming impossible for molecular biologists and biochemists to do research without the aid of computerbased tools. This module is an introduction to the concepts, and the principal databases, of bioinformatics and structural biology/chemistry. This course should enable students to access and analyze sequence and structure data, create and edit images of molecules, and present results in several formats.

Course four: Recombinant Protein Production and Applications
Instructors: Pamela Plant and Caterina Di Ciano Oliveira

In this laboratory you will learn the basics (both theory and practice) of protein purification using HIStag and GST fusion proteins as well as their downstream applications. For protein purification, you will purify the proteins from bacteria using 2 different methods; Ni2+bead and glutathioneagarose batch and column based purification and will understand the difference between them. You will learn the theory behind other tags and other purification systems and their specific applications. Finally, you will take your purified proteins, perform an in vitro binding assay as well as a RHOactivity assay and run it on a SDSPAGE and perform Western blot analysis of the resolved proteins and do a quantitation of a target band, normalized to total protein.

Course five: Adenoviral and Lentiviral Packaging
Instructor: Pamela Plant and Steve Hayes

Retroviruses are increasingly being used to deliver and express cDNA (ORF), shRNA and other constructs into either dividing or nondividing mammalian cells. The advantages in include increased efficiency of gene transfer, expression and stable integration of heritable material into the genome of target cells (using lentivirus). Packaging of replication incompetent adeno and lentiviral particles is essential to the safety of the researcher and those in their laboratories. In this course, you will learn how to produce replication incompetent adenoviruses (using QBiogene pAdEasy packaging) and lentiviruses (using 3rd generation Thermo TransLentiviral Packaging) for mammalian cell transduction. You will subclone your gene of interest into the viral vector , learn how to package, to produce and to titre your viral particles and calculate the multiplicity of infection (MOI) for a particular cell type.

Course six: Gene Expression Profiling Total Workflow
Instructor: Pamela Plant

Changes in specific gene expression patterns often reflect adaptations in cellular functions to certain biological stimuli. These transcriptional changes (in gene expression) can be assessed and quantitated using real time PCR (qPCR). In this laboratory, you will learn how to extract high quality RNA from whole tissue and cultured cells, how to analyze (using a bionanalyzer) the quality and quantity of your RNA (and why this is crucial to the success of qPCR) and then how to use this information to set up a reverse transcription reaction. The resulting cDNA will be utlilized in a real time qPCR reaction with validated primer sets. You will learn how to set up the reaction, how to validate primers (using endpoint PCR and by running standard curves) how to optimize conditions and to troubleshoot the reactions in the workflow. You will also learn indepth analysis for absolute and comparative (ΔΔCt) quantitation. Students completing this course will be competent in the total workflow needed to assess changes in specific genes using real time PCR.