Molecular Diagnostics Core

The Molecular Diagnostics research core provides investigators access to cutting-edge instrumentation for expression analysis, SNP genotyping, and other molecular assays. The available technologies include DNA sequencing, conventional and real time PCR, capillary electrophoresis and low- and high-density DNA chip workstations.

The core offers training on the use of core instrumentation, assistance in the design of experimental protocols and ongoing consultation and support. Core instrumentation is available for self-use by trained investigators. Please review the core usage guidelines below and contact Dr. Randall Olsen for more information (rjolsen@tmhs.org; 713-441-6802)

Investigators are encouraged to make recommendations regarding new technologies and services that may be useful to the research community.

Resources List

• Capillary Electrophoresis
• Spectrophotometry
• Real Time PCR
• Microarray Instrumentation
• Workstation
• Software

Capillary Electrophoresis

Agilent Bioanalyzer:
The Agilent 2100 is a microfluidics-based platform offering solutions for the analysis of DNA, RNA, proteins and cells. It answers your questions within 30 minutes, delivering automated, high quality digital data.

Location: Fondren 554 Usage guidelines: Registration on the utilization log is mandatory Users should utilize their own TMH login on the instrument computer Users should bring all disposables with them and remove after using the equipment Chips should be removed from the instrument and an electrode wash performed after use

Spectrophotometry

Nanodrop
The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of 1 ul samples with remarkable reproducibility. The patented sample retention system eliminates the need for cuvettes and capillaries, which decreases the measurement cycle time. In addition, the high absorbance capability eliminates the need for most dilutions.

Location: Fondren 554 Usage guidelines: Registration on the utilization log is mandatory Users should utilize their own TMH login on the instrument computer Users should bring all disposables with them and remove after using the equipment

Microarray Instrumentation

Affymetrix GeneChip System
The GeneChip® Instrument System is one of the most advanced microarray analysis platforms available. With the integrated GeneChip Instrument System, investigators can generate reliable, reproducible genetic analysis data. The GeneChip platform greatly improves productivity and increases the speed and accuracy of genetic studies by automating routine procedures. The flexible platform analyzes all GeneChip probe arrays for gene expression analysis and DNA analysis.

Location: Fondren 554 Usage guidelines: Users should be trained before using this instrument Users should reserve time using the Outlook calendar Users should bring all disposables with them and remove after using the equipment

Workstation

Dell Precision 490 workstation with the Affymetrix GeneChip System

Software

Affymetrix Software

• Data Collection & Management
  • GeneChip® Operating Software (GCOS)
  • GeneChip® Data Transfer Tool (DTT)
• Data Analysis
  • GeneChip® Genotyping Analysis Software (GTYPE)
  • Genotyping Console Software Version 2.0
  • Expression Console™ Software Version 1.1.1

Other Software

Partek Genomics Suite - http://www.partek.com/partekgs - Partek® Genomics SuiteTM (Partek GS) is a comprehensive suite of advanced statistics and interactive data visualization specifically designed to reliably extract biological signals from noisy data. Use Partek GS powerful statistics and interactive data visualization to easily analyze and compare a variety of genomic data sets, such as gene expression data, exon data, copy number data, mapping data, tiling data, and SNP data.

*Note: Descriptions provided by the equipment manufacturers (through website)

APPLICATIONS

Agilent Bioanalyzer
The Agilent 2100 bioanalyzer is the industry standard for RNA sample QC and has replaced labor-intensive gel electrophoresis for this application. It is also rapidly replacing gel electrophoresis for DNA fragment analysis and SDS-PAGE analysis of protein samples. A unique feature of the Agilent 2100 bioanalyzer is that it can be used for both electrophoretic separation and flow cytometric analysis of cell fluorescence parameters.

Typical applications

• RNA sample QC prior microarray or qPCR experiments
• DNA Analysis of PCR/mPCR and RT-PCR products
• Protein expression analysis
• Protein purity during purification
• Cell-based analysis in gene silencing or apoptosis

More detail on these and other applications can be found at Agilent’s website http://www.chem.agilent.com/scripts/LiteratureResults.asp?iprodinfotype=4&imodel=1115

Nanodrop

• Nucleic Acid Measurement - The NanoDrop® ND-1000 Spectrophotometer will accurately and reproducibly measure nucleic acid samples up to 3700 ng/ul, without dilution. To do this, the instrument automatically detects the high concentration and utilizes the 0.2mm pathlength to calculate the absorbance.
• Microarray labeling QC - The capability to pre-select viable fluorescent-tagged hybridization probes for gene expression in microarrays can eliminate potentially flawed samples and improve research effectiveness. The NanoDrop software facilitates the measurement of DNA concentration and dye labeling effectiveness. The NanoDrop® ND-1000 Spectrophotometer measures the absorbance of the fluorescent dye, allowing detection at dye concentration as low as 0.2 pmol/ul.
• Protein Measurement - The NanoDrop® ND-1000 Spectrophotometer will accurately and reproducibly measure purified protein samples up to 100 mg/ml (BSA), without dilution.
• Cell Culture - The NanoDrop® ND-1000 Spectrophotometer can be used to monitor light scattered by non-absorbing suspended cells. The "Cell Cultures" module displays the spectrum data from 250 nm to 700 nm. One cursor is fixed at the frequently used wavelength for monitoring microbial cell suspensions (600nm) while the second cursor can be set to the wavelength of interest.

More detail on these and other applications can be found at Nanodrop’s website http://www.nanodrop.com/nd-1000-overview.html

Affymetrix GeneChip System

• The Affymetrix GeneChip® System provides a systems biology perspective for both expression profiling and DNA analysis. This integrated system gives you the flexibility to view the genome at a global level or focus on a specific subset of genes. By integrating gene expression and DNA analysis on a single platform, the GeneChip System provides a total solution to elucidate complex mechanisms and networks underlying biological processes and diseases.
• Gene Expression Analysis - GeneChip® catalog and CustomExpress™ arrays are widely recognized as the gold standard in expression analysis. They are used by leading research labs around the world for a broad and expanding list of applications, such as: uncovering new regulatory pathways, confirming mechanisms of action, validating drug targets, classifying diseases, analyzing toxicological responses, and developing diagnostics.
• DNA Analysis - High-density GeneChip arrays enable higher resolution whole genome scans and large-scale sequencing projects, facilitating discoveries that would be difficult or impossible to make with other technologies. Whole-Genome Scanning: Complete high-resolution scans with unprecedented power in a fraction of the time required by traditional technologies. Cancer Genetics Research: Analyze regions with loss of heterozygosity at high resolution. Pathogen Subtyping: Perform complete sequence analysis of microbial pathogens to track genetic evolution and identify variations contributing to pathogenicity or drug resistance. Pharmacogenetics: Understand the molecular basis for differential drug response through whole-genome scans or target gene sequence analysis.

INSTRUCTION
All new users need to be trained before usage of equipment is allowed. Please contact Dr. Randall Olsen for more information (rjolsen@tmhs.org; 713-441-6802).

Manuals for assays performed with each instrument can be found at the manufacturer’s websites.