Determine whether a microarray experiment will advance a research aim; i.e., will large-scale gene expression analysis contribute meaningful data to a project?

Discuss the proposed project with either the Spotted Microarray Core Manager (above) or Shannon McWeeney, Ph.D., Biostatistician and Assistant Professor of Public Health and Epidemiology (503-494-8347), regarding the best way to design a microarray experiment. There are important issues regarding reference design, particularly in the ever-popular time-course studies, that should be addressed before any RNA samples are isolated.

Complete the SMC’s online project application. This form officially notifies the SMC that you are planning to start a microarray project. The form will be reviewed for a number of issues - experimental design, sample number, array type and number. Review is not perfunctory and may take a few days. We may contact you with suggestions for changes to improve your project to ensure that it will have long-term value. We will verify that we have the appropriate arrays in sufficient number for the planned experiment. If not, we will schedule a printing to make them. After we have verified that we can accept the experiment and have the necessary reagents, a project number will be issued. Please use this number in the future to refer to the project.

Run the experiment and prepare total RNA for analysis. Submit the sample information via the online sample submission form, then deliver the samples to the SMC. Basic pricing for our microarrays includes amplification, labeling, hybridization, and imaging. We are currently using linear T7 amplification for all samples. Please be aware that we have mass and volume requirements for this process. RNA must be 0.3 µg/µl and we should receive 3 µg of total RNA per sample. It is possible for us to work with smaller amounts of total RNA by doing two rounds of amplification, but there are additional charges for this.

A number of protocols are available for RNA preparation. Not all are equivalent, but we currently offer no recommendations. Our experience is that the most likely cause of microarray error is the quality of the RNA, so it is critical that the utmost care go into the preparation of the samples. Note that it is not necessary to provide poly(A)+ RNA; microarray data is generally producing using total RNA as the starting material.

The SMC will contact you when we have started working with your samples. Please be aware that, in general, it takes eight working days to go from the initial labeling to the final preprocessed data. Large projects may take longer.
SMC overview for microarray experiments

SMC overview for microarray experiments

1. Samples are labeled for Cy5 or Cy3 detection.
2. Samples are combined in appropriate pairs. Aliquots of each pair are placed
   onto each of two identical slides. M-series LifterSlips (Erie Scientific) are
   placed on the arrays, which are then placed in Deep-Well Hybridization
   Chambers (TeleChem, Inc.)
3. Hybridize overnight.
4. Batch process arrays for development of signal
5. Scan samples for Cy3 and Cy5 signal. Save as 16-bit TIFF images
6. Evaluate images
7. Grid (overlay a grid on the array image to locate the individual spots)
   and determine the average intensity for each spot using ImaGene (BioDiscovery).
8. Preprocess the data (normalization; centralization; standardization)
9. Examine data. Prepare a CD with data files and commentary.

Data will be returned to the investigators via campus mail on a CD, formatted for use in both Macintoshes and PCs. Data will be available from the SMC database after its go-live date. For data on CD, Html files are formatted to work with both Internet Explorer and Netscape, although Internet Explorer is recommended. Images are not returned to the investigator unless special arrangements are made, but are archived on tape by the SMC. Image files are 65 Mbytes each and each slide generates two, so even small projects would have multiple CDs of image files alone.

Analysis training and software are available through the Bioinformatics Suites, on the Marquam Hill Campus and on the West Campus. Bioinformatics & biostatistics support is available through the Biostatistics & Bioinformatics Shared Resource of the Center for Biostatistics Computing & Informatics in Biology and Medicine.