The DEPARTMENT of BIOMEDICAL ENGINEERING School of Engineering and Applied Science • School of Medicine
Reliable small-‐sample transcriptomics by stochastic profiling Hands-‐on Workshop August 3-‐9, 2014 Cell-‐signaling Bioanalysis Laboratory Host: Kevin A. Janes, Ph.D. Support provided by: Workshop Organizers and Instructors University of Virginia Department of Biomedical Engineering Medical Research Building 5, Room 2225 POB 800759, Charlottesville, VA 22908 Kevin A. Janes, Ph.D. 617-‐461-‐9210 (c) 434-‐243-‐2126 (o) Biography and Research interests: B.S. in Biomedical Engineering, Johns Hopkins University, 1999 B.A. in Spanish, Johns Hopkins University, 1999 La Universidad de Santiago de Compostela, Santiago de Compostela, Spain, Pharmaceutical Technology, 1999-‐2000 Ph.D. in Bioengineering, MIT, 2005 Postdoc in Cell Biology, Harvard Medical School, 2005-‐2008 Changes in cellular behavior underlie development, disease, and tissue homeostasis. The response of cells to external factors depends upon posttranslational signals and changes in gene expression. These biomolecules are wired together in cells to form networks. Intracellular signaling and gene-‐
expression networks are highly interconnected and time dependent, making them difficult to study and even harder to understand at the systems level. Our lab designs new experimental and computational approaches for analyzing such networks. We draw from engineering principles to inspire new techniques that can be applied to network-‐level questions about signal transduction and gene expression. We are particularly interested in using our methods for problems in cancer biology, where the molecular “signal processing” has gone awry and cellular responses are inappropriate. Sameer S. Bajikar, B.S. 703-‐728-‐2735 (c) 434-‐243-‐1733 (lab) Biography and Research interests: B.S. in Biomedical Engineering, University of Virginia, 2010 Ph.D. candidate in Biomedical Engineering, University of Virginia, 2010-‐present Cancer heterogeneity remains a significant challenge to the treatment of the disease. This is particularly exemplified in basal-‐like breast cancers, the most aggressive and deadly form of breast cancer. These basal-‐like tumors are often highly heterogenous in basal-‐marker protein expression, and these heterogenous tumors are coupled with an especially poor fate. However, the role that heterogeneity plays in basal-‐like breast cancer progression is unclear. Using stochastic profiling, we have uncovered a program of transforming-‐growth factor beta (TGFB) genes that are heterogeneously regulated in a basal-‐like breast cell line in 3D culture. One of these genes is growth differentiation factor 11 (GDF11), a secreted TGFB family member. Homogenizing GDF11 expression by addition of recombinant GDF11 improves 3D phenotypes in both normal and cancer breast cell lines. I am currently investigating the mechanism of action and evaluating whether GDF11 can be a therapeutic clinically for basal-‐like breast cancer. Chun-‐Chao Wang, Ph.D. 919-‐649-‐2494 (c) 434-‐243-‐1733 (lab) Biography and Research interests: B.S. in Chemical Engineering, National Taiwan University 1997 M.S. in Chemical Engineering, National Tsing Hua University 2001 M.S. in Chemical and Biomolecular Engineering, NCSU 2006 Ph.D. in Chemical and Biomolecular Engineering, NCSU 2008 Postdoc in the Janes Lab since 2008 My graduate work at North Carolina State University centered on the kinetic analysis of cellular signaling. With my background in engineering and biology, I want to do interdisciplinary research that incorporates systems biology concepts into cancer research. As a postdoctoral scientist in the Janes lab, I have been working on an extracellular matrix-‐dependent intratumor heterogeneity in cancer initiation and progression. I have dedicated effort to developing new techniques to systematically analyze regulatory heterogeneity in single cells. We have applied these methods to address important biological questions. I believe that our understanding of cancer biology would be continuously improved by new systems approaches tailored to the complexity of the oncogenic network. Lixin Wang, Ph.D. 434-‐218-‐1296 (c) 434-‐243-‐1733 (lab) Biography and Research interests: Norman Bethune University of Medical Sciences, Changchun, China, M.D. in Medine 1990, M.S. in Pathology 1995, Ph.D. in Molecular Biology 1998 Postdoc in the Janes Lab since 2008 I have been working in the basic science for more than 20 years. As an undergraduate in China, I learned medicine and did some research work in the anti-‐
tumor drug development. In the master program, I did in vitro bone marrow stromal stem cell culture and then transplanted under the mice renal capsule to observe the relationship between CFU-‐F in bone marrow and osteogenesis. In the Ph.D. program, I did bFGF molecular cloning, protein expression, protein purification and assays for the product activity. After graduation, as an employee, I did some immunology work. I constructed DNA vaccine and recombinant vaccinia virus against HIV-‐1 subtype B, and immunized mice and got promising results. After I came to USA, I started to work on DNA replication field and found some important elements that determined the origins of DNA replication. In 2009 I joined Dr. Kevin Janes lab and used in vitro 3D culture to study why only a fraction of multiacini formation upon ErbB2-‐ErbB1 dimer activation. Cheryl A. Borgman, M.S. 434-‐960-‐4957 (c) 434-‐243-‐1733 (lab) Biography and Research interests: B.S. in Botany, University of Vermont 1978 Cell Science Internship, W. Alton Jones Cell Science Center, Lake Placid, NY 1978-‐79 M.S. in Horticulture and Plant Breeding , Cornell University 1985 I have had 28 years of experience cloning, sequencing and characterizing genes from various contexts including src-‐signaling genes, genes involved in cell migration and mutant analysis of genes implicated in pancreatic cancer. My molecular biological skills have developed with the field. Consequently, I have an understanding of how and why protocols and techniques work and how to troubleshoot protocols for particular experimental designs. Recently, I have amplified 3’UTR sequence to be used for the development of a real-‐time, multiplex RNA stability assay for profiling half-‐life regulation. I have 3 years of experience sectioning and processing frozen tissue samples, which has involved significant troubleshooting due to the uniqueness and often scarcity of specific tissues. Food recommendations near the University and Downtown UVA Medical Center Corner (west on Main St from Marriott) Basil, 109 14th St – Mediterranean Lemongrass, 104 14th St – Thai Revolutionary Soup, 104 14th St NW – primarily Vegetarian and Vegan College Inn, 1511 University Ave – Greek/Italian, hot sandwiches Bodo’s Bagels, 1609 University Ave – sandwiches Boylan Heights, 102 14th St NW – sports bar, hamburgers and pub food Little John’s,1427 University Ave – delicatessen Virginian, 1521 University Ave – historic pub (opened in 1923) Christian’s Pizza, 100 14th St NW – gourmet by the piece Marco & Luca, 107 Elliewood Ave – Asian dumplings Downtown (east on Main St. from Marriott, 1 mile) Miller’s, 109 W. Main St – historic pub (where the Dave Matthews Band was formed) Orzo Kitchen & Wine Bar, 416 W. Main St – Mediterranean Eppie’s, 412 E. Main St – mixed cuisine Vita Nova, 310 E. Main St – pizza by the slice Taste of India, 310 E. Main St (downstairs) – Indian C&O, 515 Water St E – Charlottesville’s only 5 star restaurant ($$$) Stochastic Profiling Workshop Schedule Continental breakfast and coffee will be available during the morning lectures each day, lunch is available at the University Hospital cafeterias. Sunday, August 3 (MR5 -‐ Rm 2005) 8:15 Meet in hotel lobby 8:30–9:30 Welcome and introductory short talks 9:30–10 Background, motivation, and overview (KJ) 10–11 Sample preparation lecture (KJ) 11–12 Lab Tour and station set-‐up 12–1 Lunch as a group in the hospital cafeteria 1– a) Tissue embedding (CB) b) Tissue cryosectioning (KJ) c) Tissue staining (LW) d) Cell fixation and staining (CCW) Monday, August 4 (MR5 -‐ Rm 2005) 8:15 Meet in hotel lobby 8:30–9:30 Laser capture microdissection and poly(A) amplification lecture (KJ) 9:30–10 Start Proteinase K digestion (LW, KJ, CCW) 10–10:30 Trip to ID badge office (CB) 11–1 #1-‐5) Start poly(A) amplification (LW, KJ, CCW) 1–2 Lunch in the hospital cafeteria 2–5 #1-‐5) Staining and LCM (LW, KJ, CCW) 6–7 #1-‐5) Finish poly(A) amplification (LW, CCW) Tuesday, August 5 (MR5 -‐ Rm 2005) 8:30–10 qPCR lecture (CCW) 10– a) Standard curve with purified amplicon (all) b) Data analysis and interpretation (all) c) Optimization with housekeeping gene(s) (all) d) Optimization with low abundance gene(s) (all) Lunch when convenient Wednesday, August 6 (MR5 -‐ Rm 2005) 8:30–9:30 Reamplication and labeling lecture (KJ) 9:30–10:30 #1-‐5) Purify aa-‐cDNA reamplification (LW, KJ) 10:30–11 #1-‐5) Set up reamplification optimization (LW, KJ) 11–12 #1-‐5) Set up dye labeling (LW, KJ) 12–1 Lunch in the hospital cafeteria 1–3 #1-‐5) Labeling and purification (LW, KJ) 3–5 #1-‐5) Data analysis and interpretation (LW, KJ) Thursday, August 7 (MR6 -‐ Rm 2502 – note change of location) 8:30–10 Monte Carlo simulation lecture (SSB) 10-‐10:30 Monte Carlo exercises 10:30–12 Stochastic profiling analysis lecture (SSB) 12–1 Lunch in the hospital cafeteria 1–2 Stochastic profiling analysis exercises 2–3:30 Parameterization lecture (SSB) 3:30–4 Parameterization exercises 4–5 Individual game plans for 8/8–8/9 (KJ) 5– Overflow time (SSB) Friday, August 8 (MR5 -‐ Rm 2225) 8:30– Free day for independent experiments Saturday, August 9 (MR5 -‐ Rm 2225) 8:30–4 Free day for independent experiments 4–6 Results presentation and happy hour 6– Farewell dinner with the group