Saint Louis University Microarray Core Facility
Welcome to Saint Louis University Affymetrix Gene Chip Facility
The Saint Louis University Microarray Core Facility at Saint Louis University School of Medicine was established in 2006. Microarray Core Facility's mission is to provide a first class microarray service to investigators of the Saint Louis University School research community. The facility utilizes the fully integrated Affymetrix GeneChip platform for conducting gene expression studies. In this technology each gene is typically represented by a set of 11-20 pairs of probes. These probe pair sets contain pairs of perfect match (pm) probe is designed to hybridize only with transcripts from the intended gene and single nucleotide mismatch (mm) probe designed to measure non-specific hybridization. Proprietary computer algorithms are then used to compute the relative level of transcript abundance based on the composite hybridization signal from the probe pair set. Affymetrix GeneChip arrays are available for many organisms including Arabidopsis, C. Elegans, Canine, Drosophila, E. coli, Human, Mouse, P. aeruginosa, Rat, Xenopus, Yeast, Zebrafish. A complete list of chips available for gene expression profiling is listed with documentation on the Affymetrix web site.
Experimental Design: It is vital to have a good experimental design. All investigators who wish to pursue a project through the microarray core are strongly encouraged to meet with the Core Director Michael Green, Ph.D. to ensure proper experimental design and a clear understanding of sample requirements for processing through the facility including turnaround times. You can schedule a time to discuss experimental design for FREE. It is recommended that all experimental treatments be carried out in triplicates to compensate for biological and experimental variation.
RNA quality is the single most important determinant of success of a gene expression experiment. The utmost care should be taken to provide pure, nondegraded total RNA samples to the core. The Microarray Core has the following guidelines for researchers preparing RNA for microarray experiments:
- Poly-A enrichment is not necessary and does not improve performance.
- Total RNA may be prepared using any protocol. RNA prepared with commercially RNA isolation kits works well and follow the manufacturer's instructions for RNA isolation.
- If the RNA is isolated using TRIzol, RNAzol, or a comparable organic extraction procedure, we strongly recommend using a Qiagen RNeasy cleanup kit to further purify samples.
- For larger tissue samples we recommend immediately freezing with liquid nitrogen, and then grinding to a powder with a mortar and pestle. The powder can then be placed in a TRIzol reagent or other lysis buffer. Placing chunks of tissue into lysis buffer directly often leads to RNA degradation.
- RNA does not need to be treated with DNase.
- Use nuclease-free water to dissolve the RNA.
- Be sure to clearly label sample tubes with a distinct name.
- Prior to sample submission, an investigator should carefully quantitate the RNA samples.
- An investigator will need at least 500 ng of total RNA from each sample, ideally with a concentration of 50 ng/μL – 100 ng/μL.
The clients will submit samples and the following required information to the Core’s staff.
- Contact information (name, phone number, department, office/lab number) of principal investigator and primary contact.
- Research project description containing the background, purpose, and experimental design of the project and number of samples submitted.
- There must be at least three replicates of each RNA control and RNA experimental sample.
- All samples must be properly labeled with a unique identification.
- Users must also provide the appropriate Affymetrix GeneChips.
- The Core requires 500 ng of total RNA of each sample, with a concentration range of 50–100 ng/μl.
Doisy Research Center
Saint Louis University School of Medicine
Before biotinylated labeled RNA target is created; all samples quantity, purity and integrity will be accessed. RNA samples quantitation will be determined on Nanodrop spectrophotometer and quality assessment on Agilent Bioanalyzer.
- Sample Quality Check Using Agilent Bioanalyzer Service: Purity and Integrity assessment of total and mRNA samples of 25 to 500 ng/µL in concentration.
- Affymetrix GeneChip service includes:
- RNA Quality Assessment:
-NanoDrop: Total RNA concentration check
- Agilent Bioanalyzer: Total RNA integrity and purity check
-Total RNA quality check summary report
- Affymetrix GeneChip microarray processing: cDNA synthesis,IVT biotinylated labeling of target , aRNA purification and fragmentation, GeneChip array hybridization, GeneChip array washing, staining and scanning
- Visual inspection of scanned GeneChips image for anomalies and artifacts
- GeneChip array Summary: quality metrics assessment of array post scanning
- Data storage of GeneChip array data files
- RNA Quality Assessment:
- Perform preliminary statistical data analysis and generate lists showing up and down regulated genes with gene attributes.