Bart Frank, PhD
(405) 271-7258

Melissa Bebak
(405) 271-8468

Important Information:

Please contact us prior to isolating RNA for microarray experiments to discuss details of experimental design, amount of RNA needed, and pricing.  RNA is prepared by each investigator and delivered to the OMRF Microarray Facility.  RNA quantity and quality are then assessed by our staff prior to labeling, hybridization and data collection.

Commercial microarray platforms are available from  Affymetrix and Illumina. To review Affymetrix products, please click here. Information on Illumina gene expression microarrays is available here.

AFFYMETRIX
Exon & Gene Level Arrays
microRNA Expression Arrays
3’ Gene Expression Analysis Arrays

ILLUMINA
Whole-Genome Expression Beadchips for Gene Expression Analysis

RNA Requirements/FAQ’s

Amount of RNA Required for Microarray Experiments

OMRF’s Microarray Research Facility currently supports two microarray formats for gene expression studies, namely Affymetrix’s short oligonucleotide microarrays and Illumina’s long oligonucleotide microarrays.  Signal intensities vary with amounts of RNA, and the ability to detect low abundance transcripts diminishes as less RNA is used.  Suggested amounts of total RNA for each platform are listed below:

Affymetrix 3’ Expression Microarrays

Prepare a stock tube containing 10.0 µl of total RNA at a concentration of 0.625 µg/µl (6.25 µg total).  All dilutions should be made in DEPC-treated water.

Split this RNA into three 1.5 ml microfuge tubes as follows:

Tube 1:  Pipet 8.5 µl from the stock tube into tube 1 (5.31 µg).  5 µg in 8.0 µl will be used for labeling.  The remainder is required to assure a sufficient quantity for pipeting.

Tubes 2 & 3:  Add 4.36 µl DEPC-treated water to the remaining RNA in the stock tube.  Mix well and pipet 2.5 µl of this into tubes 2 and 3 (0.4 µg per tube).  These tubes will be used for quality control purposes to determine concentration by spectrophotometry and RNA quality by capillary gel electrophoresis (Agilent 2100 Bioanalyzer).

Although Affymetrix states their microarrays are capable of using as little as 1 µg of RNA, we recommend supplying 5 µg for labeling and hybridization.

Affymetrix Gene ST and Exon Microarrays

Amounts of RNA vary depending on the reagents used for labeling. Please contact us for information.

Illumina Microarrays

Please prepare a stock tube containing 10.0 µl of total RNA at a concentration of 0.1 µg/µl (1.0 µg total).  All dilutions should be made in DEPC-treated water.

Split this RNA into three 1.5 ml microfuge tubes as follows:

Tube 1:  Pipet 5.0 µl from the stock tube into tube 1 (0.5 µg).  This will be used for labeling and to assure a sufficient quantity is available for pipeting.

Tubes 2 & 3:  Pipet 2.5 µl per tube into tubes 2 and 3 (0.25 µg per tube).  These tubes will be used for quality control purposes to determine concentration by spectrophotometry and RNA quality by capillary gel electrophoresis (Agilent 2100 Bioanalyzer).

Sample Delivery

Please aliquot samples into three labeled tubes as described above.  Transfer samples to a cardboard or plastic freezer box with your name and date on the side.  Freeze samples at -80°C.  Transport the freezer box to OMRF’s Microarray Research Facility on dry ice.

Comments and Other Considerations:

Separate tubes of RNA are requested to reduce the chance of RNA degradation during multiple pipetings and/or freeze-thaw cycles.

Users should consider aliquoting multiple tubes of the same RNA for future studies such as

• Re-running a particular microarray
• Verifying microarray results with another method.  Our current quantitative PCR protocol requires 2.0 µg of total RNA to test in triplicate 40-80 experimental genes.  When sufficient RNA is available, users may wish to consider aliquoting enough RNA to confirm expression differences of 20-50 genes.

Scientists at the OMRF Microarray Research Facility are available to address technical questions about RNA isolation, labeling, and hybridizations.  Many parameters have been tested and optimized for the particular equipment in our facility.  We request that you cite our facility in publications if you find these protocols useful.  The following protocols are available for downloading.

Protocols

Succinic Anhydride Slide Blocking Protocol

Direct Cy-Labeling Protocol with Omniscript RT (modified for Ventana Discovery)

Direct Cy-Labeling Protocol with EndoFree RT (modified for Ventana Discovery)

Indirect Cy-Labeling Protocol (OMRF Modified Amersham CyScribe Labeling)

Automated Hybridization Protocol (Ventana Discovery Optimized Protocol)

Validation

Omnisript cDNA Synthesis for Quantitative Real-Time PCR (QRT PCR)

SYBR Green I Quantitative Real Time PCR (QRT PCR) Protocol

Quantitative Real-Time PCR (QRT PCR) Using EndoFree Reverse Transcriptase

Scientists at OMRF’s Microarray Research Facility are available to address questions about RNA isolation, cDNA production, labeling, and hybridizations. Many parameters have been tested and optimized for the particular equipment in our facility. We request that you cite our facility in publications if you find these protocols useful.

Frequently Asked Questions

Do you have suggestions for the design of experiment using microarrays?

How much RNA is needed for microarrays?

Which RNA isolation protocol is recommended?

Do you have other suggestions for working with RNA?

What equipment is used at the OMRF Microarray Research Facility?

Do you custom print microarrays?

What methods are used to analyze microarray data?

Send comments and questions to  Dr. Bart Frank, Arthritis and Clinical Immunology Program, OMRF

Scientists at OMRF’s Microarray Research Facility are involved in research projects in a number of diseases including arthritis, cancer, bladder diseases, liver diseases, sexually-transmitted diseases, and ophthalmic diseases, as well as advances in bioinformatics.  Some of these projects are directed at a basic understanding of the disease process, while others are designed to monitor the progression of the disease and response to therapy in particular patients.

Peer-Reviewed Microarray Publications from the OMRF Microarray Research Facility

The Microarray Research Facility is located within the Oklahoma Medical Research Foundation on the first floor of the Acree-Woodworth Building at 825 NE 13^th Street.

Researchers may contact us by e-mail or telephone to discuss the application of microarrays to your research.

The Microarray Research Facility Team

Bart Frank, Ph.D.
Research Associate Member
(405) 271-8468
Mail Stop 58

Melissa Bebak
Research Assistant
(405) 271-8468
Mail Stop 58

Note: Users are encouraged to contact the staff at the OMRF Microarray Research Facility to discuss details of experimental design prior to isolating RNA for microarray experiments.  For more information please see our Services tab.