Our Core Facilities and Equipment
- Molecular Biology Core
- Genomics and Bioinformatics Core
- Histology and Bio-Imaging Core
- Immunology Core
- Biochemistry Core
- Animal Research Facility Core
The Molecular Biology Facility at Arkansas Children’s Nutrition Center caters to the needs of ACNC investigators with the mission of providing cutting-edge molecular techniques in addressing specific experimental issues. The facility is overseen by Dr. Kartik Shankar. The facility, as a whole, works closely with each investigator and also, at times, integrates the expertise of other centralized facilities in the ACNC such as the Cell Culture Core and the Histology Core. The Molecular Biology Facility offers a multitude of specific services and technical expertise and training in a variety of techniques. Some examples include Southern blot and cloning of genes of interest into expression vectors for either stable or transient expression in mammalian cell culture. This core is especially well suited for studies of nutritional and developmental regulation of gene expression, pathway analysis, and gene expression profiling during various physiological and pathological states.
The facility also houses two real-time RT-PCR instruments (iCycler, BioRad labs and Agilent Prism 7000 Sequence Detection System) for gene expression analyses. In addition, the facility assists investigators in primer design (using Primer Express software, Agilent Technologies) for regular PCR or real-time RT-PCR, and primer design for other PCR applications such as cloning or regulatory sequences of genes. The Molecular Biology Facility also has four thermal cyclers (two ABI GeneAmp 2700, one ABI GeneAmp 9700 and one Eppendorf Mastercycler) for PCR applications. The molecular biology facility also is outfitted with necessary equipment to perform gene expression analyses using Northern blots, RNAse protection assays, in vitro transcription and nuclear run-on assays. Dedicated apparatus and bench-space are available for use of radioactive isotopes (α or γ-32P or 33P-nucleotides). In addition, the laboratory facilitates assessment of DNA using Southern blot, and protein-DNA interaction using electro-mobility shift assays. The Molecular Biology Facility also assists in performing luciferase-based reporter assays using the MLX Microtiter Plate Luminometer. Equipment to perform immunoblotting is available. Further, the facility has a BioRad Chemidoc XRS, BioRad GelDoc 1000, GS-700 Imaging densitometer, ImageScanner III, Benchmark microplate photometer, Personal Fx molecular phosphorimager and Kodak SRX-100 automated film developer, along with several data acquisition and analyses software.
The ACNC Genomics and Bioinformatics Facility, a designated Affymetrix core facility, is equipped with an Affymetrix GeneChip Fluidics 450 platform, hybridization oven, GeneChip 3000 scanner and Dell Optiplex PC workstation with Affymetrix Command Console Software. In addition, other instrumentation required to perform all the steps of microarray analyses from RNA extraction, purification, quality control analyses, cDNA synthesis, cRNA synthesis, labeling, hybridization and washing are part of the ACNC Genomics and Bioinformatics Facility. These include, polytron homogenizers, refrigerated table-top centrifuges, Agilent 2100 Bioanalyzer, BioRad Smartspec plus, BioRad Experion automated electrophoresis station, Nano Drop ND-1000, spectrophomoters, thermal cyclers, PCR hoods, heat-blocks, incubators, etc. Dedicated computers are available for data analyses and bioinformatics of microarray data. Centralized access and training for GeneSpring GX (Agilent Technologies), GCOS, Ingenuity Pathway Analyses, Pathway Studio, Cluster, TreeView, GenMapp, MS Excel and other software are available through the facility. The ACNC Genomics and Bioinformatics Facility is state-of-the-art and is capable of examining whole genome expression (human, rat, mouse, porcine, etc.) arrays and newly emerging technologies such as ChIP-on-chip, methylation arrays and tiling arrays.
The Histology and Bio-Imaging Facility assists in the routine processing of tissues from experimental studies for histological and immunohistochemical analyses. The facility has an automated Sakura Tissue Tek Vacuum Infiltration Processor for processing tissues and a Tissue Tek Embedding platform. The facility also has a Leica RM2135 microtome for cutting paraffin or acrylic embedded blocks. The laboratory utilizes a Microm HM 550 cryostat for performing analyses on frozen sections. The facility has standardized protocols for histochemical and immunohistochemical staining that can be done manually or performed using an automated Dako autostainer. The facility is staffed with certified histo-technicians. Another function of the facility is to fulfill the bio-imaging and image analysis needs of the investigators. The facility has an Olympus BX50 upright microscope and a Nikon SM2800 Sterioscope fitted with a Sony DXC970MD 3-CCD camera. This workstation is also equipped with a Dell Precision computer with MCID Elite software. The MCID Elite software is complete with histomorphometry capabilities, density scanning and 3-dimensional reconstruction and rendering capabilities. In addition, the facility has an Olympus IX71 Inverted Fluorescence microscope equipped with Osteomeasure software. This system is set-up to perform both static and dynamic histomorphometric analyses of skeletal samples. Further, light and fluorescence imaging needs of investigators are met using the Zeiss Axiovert 200M Inverted microscope. This microscope has a motorized stage, z-stack capabilities, and can read DAPI, Alexa 488 and Rhodamine dyes. It is also equipped with AxioVision software which is capable of complex histomorphometric analyses on acquired images and can be used on entire batches of images. The Histology Facility also has general laboratory equipment necessary for effective functioning including slide warmers, ovens and incubators dedicated for histological procedures. The facility acts as an important resource for training, supervision and development of new immunohistochemical procedures.
The Immunology Facility is overseen by Dr. Shanmugam Nagarajan. The facility houses 14 CO2 incubators, 5 NuAire Laminar Airflow hoods, a Nikon TMS microscope and a Ziess Invertoscope. The facility also has a centralized cryostorage repository for storage and archiving of cell culture stocks. The facility offers introduction, handling, propagation and maintenance of various cell cultures. In addition, the facility has a BioRad Genepulser II and an Amaxa Nucleaofector II for electroporation of exogenous plasmid vectors or siRNA into specific cell lines. The facility also has a BD FACS Calibur Flow Cyometer and cell sorter for quantitative assessments of different cell populations. Assays for cell proliferation, apoptosis, viability, oxidative stress and presence/absence of cell surface antigens are routinely performed on this instrument. In addition, the Immunology facility has a BioPlex multiplex bead array system. The Bio-Plex system utilizes Luminex xMAP technology which permits the multiplexing of up to 100 different assays within a single sample.
The Biochemistry Core is overseen by Dr. Martin Ronis. The Core has experienced staff who conduct routine biochemical and endocrine analyses as a service to the rest of the ACNC. The Biochemistry Core is responsible for conduct of enzyme assays for phase I and II metabolism of drugs, carcinogens and steroids using spectrophometric, spectrofluorimetric, TLC, GC-MS, HPLC and radio-HPLC assays. Substrate-specific assays for all the major cytochrome P450 enzymes, sulfotransferases, GSTs and UDPGTs are standardized. Routine analysis of protein content, intermediary metabolism, lipid peroxidation and GSH/GSSG are conducted by spectrophometric assays and by HPLC-utilizing electrochemical and photoarray detection. Metal analysis is conducted using atomic absorption spectroscopy. The Core runs quantitative Western immunoblot analysis for enzymes, transcription factors and other cellular proteins using antisera obtained from collaborators and commercially available antibodies and characterizes peptide-specific antisera made under subcontract. The Biochemical Core conducts routine analysis of hormones and cytokines by radioimmunoassay and ELISA using commercially available kits and NIH supplied reagents for such hormones as GH and LH. In addition, protein purification of enzymes and transcription factors is conducted by the Biochemistry Core.
The laboratories are equipped with two Shimadzu Scanning Spectrophotometers, a scanning Spectrofluorimeter, HPLC systems with auto-samplers and MS, UV, Photodiode Array, Coularray and radioisotope detectors. In addition, there is a GC with flame and electrochemical detector and a GC-MS with both head space and liquid auto-samplers. SDS-PAGE apparatus are available in MiniGel, medium and large sizes and there is a bench top X-ray film developer. In addition, the ACNC possesses a BioRad Molecular Phosphoimager and Densitometer capable of quantitative densitometric analysis of X-ray film, direct phosphoimaging of 32P, 35S, 14C and 3H signals on gels, nitrocellulose filters and TLC plates and chemiluminescence. There is also a Shimadzu Atomic Absorption Spectrometer for analysis of metal ions, a gamma counter for quantification of radioimmunoassays and an FPLC protein purification system.
The Animal Research Facility (ARF) at Arkansas Children’s Nutrition Center is directed by Dr. Thomas Badger and conducts all studies involving live animals. The facility consists of a team of highly trained technical staff which includes 7 full-time animal care personnel. The facility is part of a new state-of-the-art AAALAC approved animal facility at Arkansas Children’s Hospital Research Institute (ACHRI). The ACHRI animal facility has an additional staff of 10 individuals including board certified veterinarians, an animal facility manager and animal care personnel who work in close conjunction with the ARF team in conducting basic animal husbandry services. All aspects of executing ACNC animal studies are carried out by the ARF staff, including starting IACUC protocols, tracking animals (using subcutaneous microchips, eartags, etc.), performing serial in vivo assessments (e.g., body composition, indirect calorimetry, insulin clamp studies, oral glucose tolerance tests, and peripheral quantitative computerized tomography, pQCT) and collection and banking of tissues. Surgical manipulations such as intragastric and intravenous cannulations, implantations of infusions pumps, and surgical resections of ovaries, pituitaries, liver, etc. are also regularly carried out by the ARF staff. The ARF also has facilities assessment of whole animal imaging and body composition using X-ray computerized tomography, nuclear magnetic resonance and dual x-ray absortiometry. Specific analyses of bone mineral density, content and other skeletal parameters can be carried out in vivo (rodents) or ex vivo (in rodents and pigs) using pQCT within the ARF. Facilities to assess blood pressure using a tail-cuff in rats are also available and routinely employed by investigators. In addition, a separate insulin-clamp lab is available for investigators to perform experiments under euglycemic or hyperglycemic conditions. The ARF is also equipped with a Comprehensive Laboratory Animal Monitoring System (CLAMS) to perform 24-hour metabolic assessments of food and water-intake, indirect calorimetry and activity measurements. These assessments can be carried out in rats and mice. The ARF conducts experiments with rodents and other large animals such as pigs. Separate barrier housing for transgenic and knockout mice are also available. Data recording and archiving are performed according to standardized protocols developed by the ARF and all data are maintained through databases specifically designed to meet particular needs of each animal study.