Welcome to the frequently asked questions (FAQ) list for the MicroArray Forum. In order to keep the Forum manageable, we will periodically extract and compile questions from the Forum and add them to the FAQ - please check this document before posting!
Contributors to the FAQ:
Joe DeRisi (JD)
Kathleen Hayashbara (KH)
Michael Eisen (ME)
Paul Spellman (PS)
Vishy Iyer (VI)
Questions concerning this document may be addressed toTae Bum Shin. Questions about microarrays not covered by the FAQ should be posted on the Forum.
(1/20/99) FAQ created and posted - all entries are new!
I. General Questions
1. What are some good references about microarrays and its applications?
There are several papers explaining the principles and applications of DNA microarrays. For a good start, check out the Stanford Genomic Resources website ( http://www-genome.stanford.edu ) to find a list of these papers and their supplementary material.
II. Microarrayer Construction
1. How do I make my own microarray printer?
- See the MGuide for the instructions and the latest software
2. I'm not working in the U.S. Can I order the necessary parts to build a microarrayer?
- We know of groups in Asia and Europe that have ordered and assembled their own microarrayer. Some the suppliers may have local distributors in your area, so check with them.
3. What other groups have built a microarrayer?
- Several other sites have generously provided WWW links to their sites to show off their results.
Albert Einstein College of Medicine ( http://sequence.aecom.yu.edu/bioinf/funcgenomic.html ).
III. Slide Preparation and Printing
1. What types of printing tips are/have been used?
- See the tip gallery for pictures and comments about all the tips we have tried.
2. How do you remove excess solution from the printing tips? The first several slides were ruined due to the large drops.
- The area next to the 96 well plate is reserved for a blot pad, an extra slide for depositing the excess liquid. The arrayer can be programmed to tap this blot pad ten times before printing on the remaining slides.
3. What other slide-coating substrates can be used?
- Poly-Lysine is still our recommended coating material for slide preparation. Experiments have been tried with aminoalkylsilanes, but the DNA binding capacity seems to be much lower and it appears to be more susceptible to fluorescent impurities. Please let us know of any successes that you might have with alternate coating materials. (KH)
4. What humidity conditions are required during printing?
- We generally keep our printing room at a slightly higher humidity (~50%), but this does not seem to be necessary during printing. However, it does reduce evaporation from the 96-well plate.
5. How does the tip cleaning procedure work?
- The tips are cleaned by two cycles of washing and drying. Most of the excess liquid is removed from the tips by ddH2O, followed by drying with a vacuum-induced airflow. To ensure quality when printing with 16-tips, a regular sonication may be required after each plate, as well as a closer fit with the drying station ports.
6. After printing, can I verify the microarray slides for the presence of DNA?
- There is no efficient way to do this short of an actual hybridization. However, we have found that observing the dried salt residue on the slide is an excellent indicator of array quality. Also, the absolute amount of DNA at each array element is not important, since analysis of the signal at each spot involves measuring the ratio of two probes. (VI)
7. Why do the slides need to be aged before printing?
- It was discovered that "aging" of the slides improved the signal-to-noise ratio of the probe hybridization. The actual mechanism of aging process is not yet understood.
8. During the slide post-processing, can an 80°C oven be used instead of UV crosslinking?
- We have not tried this alternative, although in some cases we have found that even the UV crosslinking may not be necessary.
9. Can we recycle the poly-lysine solution?
- We don't recommend recycling the poly-lysine, particularly in light of the importance of this step.
IV. Probes, Dyes, and Hybridization
1. What's the best method to work-up the PCR reactions?
- Isopropanol-mediated precipitation has been shown to be the best method. Use 96-well V-bottom plates, and resuspend the precipitate in 15-20uL 3xSSC. The concentration of each sample is not critical nor does it have to be the same - most of the printed DNA is washed away during the post-processing, leaving a thin monolayer. (ME)
2. Why don't the O-rings fit in the hybridization chamber?
- The O-rings are exactly the correct size. Don't stretch them prior to or during their installation.
3. What is the CyDye incorporation rate during the cDNA labeling?
4. Are there differences in the incorporation rates of Cy3 vs. Cy5 dyes?
- Although there may be differences, these will be removed during the normalization of the scanned images during analysis.
5. Can we perform the PCR reactions in 384-plates?
- Although the microarrayer can print from 384-well plates, using these plates for the PCR reactions have been reported to be inconsistant with respect to quality and yield. The best method is to use a 96-well format PCR, and transfer the precipitated product to 384-well plates for printing.
6. Can we recover the CyDyes after labeling? It seems like a waste to throw out the remainder.
- Yes! See our protocol for HPLC purfication of CyDyes.
7. How are the PCR primers designed to avoid cross-hybridization?
- All of the PCR products are the complete ORF sequences (although some are only partial ORFs). Hence any cross-hybridization will be due to real sequence homology or repetitive elements.