http://uscnorriscancer.usc.edu/Core/MolGen/

The Molecular Genomics Core (MGC) offers high-throughput analyses of genetic, epigenetic and transcriptomic variation. The MGC was created in the summer of 2009 through the merger of the DNA Core, the Microarray Core and the Genomics Core. The MGC is divided into an HSC (Health Science Campus) component and a CHLA (Children’s Hospital, Los Angeles) component.

Major Services, Technologies, Major Equipment and Expertise Provided

Consultation. Users are encouraged to meet with the core director or co-director, as appropriate, prior to project initiation to discuss their needs for the proposed project. The goal of this consultation is to educate the core about the researcher’s needs and to educate the user about possible approaches and services.

Direct Services. Direct services provided by the MGC include sample preparation and analytical assays. Sample preparation is performed on research samples using good laboratory practices or from clinical samples in a CAP/CLIA-certified laboratory for clinical research and diagnostics.

Sample preparation. Biospecimen processing is available for research (HSC) and clinical (CHLA) samples from body fluids, fresh and archival tissue, and non-mammalian sources (i.e. plasmid/phage DNA for capillary sequencing).

  • DNA isolation. High-throughput DNA isolation from blood, buffy coat, buccal cells (swabs and mouthwash), saliva, serum, semen and paraffin-embedded tissue is available through the core. A majority of the DNA isolation chemistry utilizes magnetic bead-based isolation kits available to run on the Maxwell Particle Movers using < 400 ul of sample. All purified DNA is transferred into either 1D or 2D barcoded tubes for storage and is typically divided into at least two aliquots for storage in separate containers.
  • NA isolation. The core encourages users to collect specimens in specially designed containers to stabilize source RNA at the time of collection for whole blood (PAXgene RNA Blood Vacutainers), from saliva collections (RNAprotect Saliva Reagent), or from solid tissue (RNAlater Tissue Protect Tubes). For formalin-fixed, paraffin-embedded (FFPE) samples, blocks are resectioned, a pilot section is stained, and a representative area is chosen by a consulting pathologist that is then cored with a 2 mm punch. The extracted core is immediately placed in RNA extraction medium for further processing
    [Maxwell 16MDx, QIAsymphony or manual (Agencourt)]. Samples previously collected or collected in containers that do not preserve the RNA at time of collection are processed based on the type of tissue submitted. Isolated RNA is stored at – 80°C in 1D or 2D barcoded tubes.

Analytical assays. The analytical assays available are based on the ability to identify and interrogate genetic (DNA targeted profiling) and epigenetic variation (epigenetic targeted profiling). Expression profiling encompasses both genetic and epigenetic components. For example, direct changes to the DNA sequence can have effects on gene expression levels, and DNA methylation and histone modifications also play a role in modifying gene expression levels through changes to chromatin confirmation. The core offers users the ability to obtain several levels of throughput on the three categories of analysis (DNA profiling, epigenetic profiling, expression profiling) depending on needs.

  • DNA profiling. The basic unit of analysis for DNA profiling is a single base in the genome. Low-throughput analysis encompasses the measurement of single bases one at a time or a few in parallel. Single nucleotide polymorphisms (SNPs) can be assayed using a modified 5’ nuclease assay (“TaqMan” assay) with fluorogenic probes that are specific to one of the two alleles present at the SNP. Parallel analysis of multiple custom SNPs is possible from as few as 48 SNPs to several thousand SNPs. Intermediate analysis of DNA provides genome-scale coverage while sampling a subset of the genome. The services offered in this category are genome-wide SNP chips that attempt to provide genome-wide coverage through the use of correlations between the assayed SNPs and known common variation in the genome. In addition, the core offers analysis of single DNA clones using capillary electrophoresis DNA sequencing through our outsourcing partnership with Genewiz.
  • Epigenetic profiling. Low-throughput analysis of DNA methylation is performed using a modification of the TaqMan procedure MethyLight, which is quantitative over four orders of magnitude, highly sensitive and requires only post-PCR data analysis without manipulation. In addition, bisulfite DNA sequencing is available for evaluating the methylation status of CpG dinucleotides from individual subclones from a target region. This methodology allows for haplotyping of methylation across a small region of DNA (400-800 bases). Intermediate-throughput analysis of DNA methylation is provided using Illumina Infinium Methylation chips. The current chip (HumanMethylation450) is a panel of assays to interrogate the DNA methylation status at 482,421 CpG loci located within the genome. The core has extensive experience with this product, serving as a Genome Characterization Center (GCC) as part of The Cancer Genome Atlas (TCGA) project.
  • RNA profiling. Low-throughput RNA profiling is available using the Fluidigm Biomark system on 48 or 96 targets in parallel using qPCR or up to 800 targets in parallel using the Nanostring nCounter system. It provides a direct measure of signal based on the capture of individual RNA molecules and a digital measure of expression levels. In addition, the core offers services for intermediate-throughput RNA profiling using a series of fixed content arrays from both Affymetrix and Illumina. These arrays offer genome-wide coverage of a reference panel of known expression targets that represents current knowledge of the transcriptome.

Investigator operated services. A variety of core equipment is available to users after training. Nanodrop, Bioanalyzer and Experion QC platforms are available, with users covering the cost of reagents. Users also have access to a Tecan EVO robot with 96-well fixed-head for sample plating, the Fluidigm Biomark system for qPCR and SNPs, an ABI7900 real-time PCR system and a Covaris S2 Ultrasonicator.