Nucleic Acid Extraction, Quantification, and Quality Control (QC)
- RNA and DNA extraction services are available for cells, tissues, biofluids, and FFPE. Where required sample homogenization can be carried out using cryo-fractionation (Covaris CryoPrep) or grinding (Miltenyi GentleMACS) in sealed single-use vessels. Our standard extraction procedures use Qiagen kits. Other commercial protocols may be available upon request. Phenol-Chloroform extraction is not available.
- Sample quantification is available using Qubit or Quant-iT dye-based systems or micro-volume spectrophotometry using NanoDrop.
- Fragment analysis, including services to determine RNA integrity or high throughput genotyping are available using Agilent BioAnalyzer 2100 or an AATI twelve capillary Fragment Analyzer. Both systems are capable of analysis of low or standard input DNA or DNA samples (~1 - 100 ng) with fragment sizes up to about 10 Kb. Neither system is suitable for assessing the integrity of genomic DNA (gDNA).
Gene Expression and Molecular Quantification Assays
- The nanoString nCounter® platform is a complete solution for detecting and counting large sets of molecules using solution-phase hybridization, immobilization and fluorescence scanning. Digital molecular barcoding enables researchers to directly count the number of target transcripts in each sample. nanoString barcodes enable multiplexing and can quantify up to 800 RNA, DNA, or protein targets per sample. The OGSR runs an nCounter Gen2 Analysis System designed for core facilities and can operate at a throughput of 24 samples per day.
- We provide ddPCR services on Raindance Technologies RainDrop and BioRad QX200 systems in the OGSR. ddPCR is particularly suitable for applications such as genotyping or validating somatic mutations, viral load counting and absolute transcript quantification. Assays are typically run using hydrolysis probes (Taqman) and can be multiplexed using dual channel detection. Validated hydrolysis qPCR assays can usually be transitioned easily to ddPCR.
- miRNA qPCR services are available using Qiagen/Exiqon LNA primer technology. We offer services for all consumables compatible with our Roche LC480 384-well platform. 96-well assays are not available.
Next Generation Sequencing (NGS)
Investigators at the University of Miami can access NGS services on campus at both the OGSR and the John P. Hussman Institute for Human Genomics, Center for Genome Technology (CGT). OGSR services support Sylvester members requiring fast turnaround for lab-scale projects.
- For transcriptomics, the principle service we provide is stranded total RNA library prep with ribo-depletion. This is suited to examining non-polyadenyalted eRNA and lncRNA expression, as well as, gene expression and pathway analysis. We also provide mRNA-Seq services for poly-adenylated RNAs. RNA cancer genotyping is available using the Illumina PanCancer RNA panel.
- Library prep for fragmented DNA to support ChIP-Seq is our principle DNA sequencing workflow. This service can also support aptamer library sequencing. DNA library prep for samples requiring fragmentation is also provided and is suited to the production of long-amplicon libraries. Services for targeted sequencing of cancer hotspots are also available. Bulk sample T and B cell receptor repertoire sequencing is supported using ArcherDX Immunoverse assays. This approach provides un-paired TCR/BSR sequencing.
- Aside from in-house library prep, the OGSR provides a sequencing only “walk-up” NGS service, whereby user can provide their own NGS libraries or library pools for sequencing.
Single Cell Genomics
- A 10x Genomics Chromium single cell controller that enables the study of thousands of cells pre run has recently been purchased and is now additionally supported by sample QC on a Nexcelom K2 imaging cell counter. Services are currently available for 3’ and 5’ gene expression and T cell and B cell receptor repertoire sequencing.
- The OGSR also has access to a Fluidigm C1 system via the Miami Center for AIDS Research (CFAR). Index FACS-based 3’ whole transcriptome analysis (3’ WTA) is available in partnership with the Flow Cytometry Shared Resource (FCSR).