NCI Program Project in Esophageal Carcinogenesis
Core D - Molecular Biology/Gene Expression Core
|Director:||Gary D. Wu, M.D.|
|Technical Director:||Sue Ann Keilbaugh, Ph.D.|
The Molecular Biology/Gene Expression Core (MBC) provides four critical services to support the scientific goals of this Program Project, namely: A) Molecular Reagent Bank; B) Image Analysis and Gene Expression Quantification; C) Genomic Services and D) Esophageal Cell Line Bank, all of which are focused towards esophageal cancer research:
Molecular Reagent Bank:
Each of the Program Project Leaders and their personnel have access to specific plasmids, expression constructs, reporter constructs, retroviral/lentiviral/adenoviral vectors and RNA/DNA isolates that are relevant to the study of esophageal biology as it relates to carcinogenesis. These reagents have been organized and centralized in the MBC. Any new reagents will be added to the bank as generated. This provides the P01 investigators archival alarmed storage of molecular reagents specific to the P01. Nearly all the reagents provided by this bank are unique reagents that are not available commercially. A general list of molecular reagents is available to all investigators via the Core website or the link below.
P01 Molecular Reagents (Excel):
Molecular Biology/Gene Expression Core website: http://www.uphs.upenn.edu/moleculr/Scientific_Cores/Molecular_bio.html
Image Analysis and Gene Expression Quantification:
The Image Analysis and Gene Expression Quantitation section of the Molecular Biology/Gene Expression Core is housed on the 9th floor of BRB. Multiple instruments are provided by the Core for use by P01 investigators to analyze images, quantify DNA/RNA/protein, isolate genetic material and perform transcription reporter gene assays. The Core not only provides oversight and support for service contracts for each of these instruments, but also provides training in the use and interpretation of raw data generated from each instrument. The Core also provides several software programs: Primer Express for the design of primer probe sets for use in kinetic PCR, Prism 4 (Graphpad) for statistical analysis and graphing, MacVector 7.0/DNAstar 5.0 (Lasergene) for DNA/RNA for sequence analysis and Partek Genomics Suite 6.4 for microarray dataminig. A list of the more heavily used instruments in the Core is noted below. Those instruments which have an asterisks (*) after them can be reserved for use by an on-line reservation service through the Core or the Flow Cytometry Facility, depending on the instrument (http://somapps.med.upenn.edu/molecular/Molbio/mrbs/day.php?
year=2013&month=08&day=29&area=2&room=1 for reservation of QPCR instruments).
Molecular Dynamics 840 Phosphorimager
BioRad GelDoc XR+
Promega GloMax Multi Detection System (luminometer)
Kinetic PCR Thermocycler (ABI 7000)*
Real-Time PCR System (Step-One Plus, ABI)*
Odyssey Infrared Imaging System (Li-Cor)
Thermal Cycler (ABI 2720)*
Dissecting Microscope (Olympus)
Nanodrop 2000 (Thermo Fisher)
FACS Calibur Flow Cytometer* (collaboration with Flow
Cytometry and Cell Sorting Resource – see Charles H.
Pletcher, Jr., Technical Director, for training and access)
The ability to examine and quantify alterations in gene expression is fundamental to the understanding of molecular mechanisms that determine biological processes. This section of the Molecular Biology/Gene Expression Core is made up of three components which address this need for the P01: 1) Microarray Gene Profiling, 2) Next Gen Sequencing Core (NGSC, Penn), and 3) Bioinformatics (NGSC and DFCI).
- Microarray Gene Profiling. The Core collaborates with the Penn Molecular Profiling Facility (https://somapps.med.upenn.edu/pbr/portal/microarr/) under the direction of Dr. Vivianna Van Deerlin (Director) to provide Affymetrix microarrays for gene expression profiling. The profiling facility also supports the use of other microarray platforms such as Agilent and Illumina as well as assays for Sequenom, Luminex, Fluidigm and TaqMan. Advice on experimental design, methods of RNA isolation and quantification are available in the Profiling Facility. Since there are many large publically available transcriptome datasets relevant to esophageal carcinogenesis, the MBC will continue to support the use of microarrays for gene expression profiling by the Penn Molecular Profiling Facility. P01 investigators are eligible for priority access to the Affymetrix systems.
- Next Gen Sequencing Core. Advanced sequencing technologies are available to P01 investigators. The MBC will subsidize, up to $4,000/year, the use of services provided by the Penn Next Generation Sequencing Core (NGSC) under the direction of Dr. Klaus Kaestner. The NGSC offers ultra high throughput sequencing services for the PSOM research community including library quality assessments, sequencing, and preliminary data analysis for a wide variety of experimental protocols including ChIP-seq, RNA-Seq, HITS-CLIP, miR-Seq, exome capture, and whole genome shotgun bisulfite sequencing. There are limited library preparation services, but the NGSC can advise on library preparation techniques. The NGSC provides the following services: 1) Experiment planning; 2) Training in library preparation; 3) Library quality assessment; 4) Sequencing and scheduling; 5) Data quality assessment; 6) Data sharing; 7) Data analysis services; 8) Data submission to public repositories; and 9) Data archiving. Please see the NGSC website for further information: http://ngsc.med.upenn.edu/.
- Bioinformatics. The NGSC will provide an initial analysis of sequencing data, however, customized analyses are beyond their scope. Intensive bioinformatic analyses focused on the specific needs of the P01 investigators are provided by the MBC through Core support of a computational biologist, Dr. Shouyong Peng who is currently a member of the Wong and Bass labs at the Dana Farber Cancer Institute. Dr. Peng is experienced specifically in esophageal cancer bioinformatics, which provides an outstanding opportunity to accelerate genome-based studies for the P01 investigators.
Esophageal Cell Line Bank:
Observations of gene expression patterns based on the use of cell culture lines and model systems need to be validated in tissue obtained from humans. The Molecular Biology/Gene Expression Core, in collaboration with the MPIC, routinely obtains human tissue from both normal and diseased esophageal tissue for the following:P01
- Esophageal RNA/DNA Bank. RNA/DNA is isolated from snap frozen tissue for use in gene expression studies (for example, Northern blots, RPA, RT-PCR, gene arrays, RNAseq, CHIPseq). The quality of each RNA sample is determined by gel electrophoresis as well as by Northern blot or RT-PCR to confirm tissue-specific gene expression.
- Esophageal Cell Line Bank. Over the past 5 years, P01 investigators have acquired, developed and utilized a large number of primary, immortalized and tumor-derived esophageal cell lines for studies. These cell lines are of both murine and human origin. The tumor-derived cell lines are named TE (#1-15), HCE (#4 and 7), TT and TTn. The primary murine esophageal cell lines are MEK3N and MEK3N whereas those of human origin are named EPC1, EPC2, and OKF6. The Molecular Biology/Gene Expression Core maintains an Esophageal Cell Line Bank. This provides a backup, centrally located, alarmed liquid nitrogen storage system to archive these cell lines for the P01 investigators. A list of the cell lines in the Bank can be accessed below. Details about the cell lines are available through the Esophageal Wiki.
P01 Esophageal Cell Lines (Excel):
Esophageal Wiki (password protected): https://weblogin.pennkey.upenn.edu/login?factors=UPENN.EDU