Updated (1/20/99)

Questions concerning this document may be addressed to Tae Bum Shin. Questions about microarrays not covered by the FAQ should be posted on the Forum.

1. What are some good references about microarrays and its applications?

There are several papers explaining the principles and application 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.

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 both in Asia and Europe that have ordered and assembled their own microarrayer. Some the suppliers may have local distributors local 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 ).

1. What type of tips are 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 verifying the print quality by observing the dried salt residue on the slide is an excellent indicator of 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.

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 for the hybridization chamber fit?

- 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?

[pending]

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 the PCR reactions be done in 384-plates?

- Although the microarrayer can print using 384-well plates, PCR reactions at this scale 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.

1. Are there commercial sources for DNA microarrays?

#113 What companies would you recommend for purchasing the sonicator? Are there any

specifications (gauge, etc.) for the additional wires used to extend the z-stage limit switches?

Do jumpers (connecting VCC and LSCOM on the ICM-1900) come in different sizes? If so,

what do I need for this particular job. Thanks, David

#115 Dear Madam or Sir, I got the prices of every part of an arrayer, but i really want to if i can get

whole one from some where? And how about the price? Thanks in advance. Best regards Bian

Xuelin

=>#118We recently purchased our second arrayer from Genemachines in Redwood City, CA. We are

quite happy with the product. Check out their website at www.genemachines.com. Their

pricing ranges from 50 - 75K and that includes all of the hardware and software. If you're

interested, call them and ask for Scott Smith. Good Luck!

#116Hi Arrayers, does anyone now a free or inexpensive software package for analysis of

Microarrays ? My image files are in GEL-format derived from a MolDyn SP Phosphorimager.

I can also convert them to TIFF format. Where is the current Website of the ScanAlyze

Software? I tried the links in this forum, but they dont work anymore. Which (also more

expensive) software can you recommend for image processing ?

#119 Is there a "latest" version of the Cy3 and Cy5 cDNA synthesis protocol? I saw the protocol in

the Science'97 article. I've seen other protocols that use Cy3 or 5 -dCTP instead of dUTP.

What's the difference?

#121 I have seen the printing tip gallery with all the tips you have tried over the years. At this point,

what tip would you recommend considering frequency of breakage, spot consistency,

difficulty in cleaning, price, etc. ? Thanks in advance for answering these questions, as well as

the ones I sent earlier regarding the building of the arrayer. David

#123Can you give me an estimate of how many man hours would be involved in the assembly of a

micro array machine? Assume that all of the parts are available and the person(s) doing the

assembly have mechanical/electrical backgrounds. Thanks.

#128 Some comments for others. Re: Assembly time. The MGuide says that they have completely

assembled it in 48 hours. I think I might be able to do my second one in that amount of time,

but the first time it took me more like 150 hours. There were a number of problems I ran into

that took a bit to figure out, just like building any system. (For instance, a pin got bent on a

connector, and shorted out a $2.50 buffer chip. It took quite a while to track down that

problem and about 10 minutes to fix.) The mguide instructions are very good follow them

closely and it will save you time. Re: Analysis software Scanalyze seems to be the standard

analysis software. It's at http://rana.stanford.edu/software/ScanAlyze.zip (See post #77) Also,

if anyone has the format for .scn files (the one's scanalyze reads), could you post it. It would

save me some time figuring it out. It appears to be a header followed by the image data (16

bits/pixel) but it's not clear to me what exactly the format of the header is. --Jim

#129 Does anyone know anything about a supposed microarray biochip meeting in Miami in

February of 1999?..and a very basic question...in the Brown lab protocols for slide

preparation it mentions a metal slide rack..could someone please tell me where i might find one

of those.....someone has to ask the easy ones....thanks..shanna

#130 This is in reply to Lance Miller's entry of 10/25/98. We have twice seen a result perhaps

similar to one you mentioned, using PLL coated slides, ie. the scanned slide was "covered

with a non-uniform background fluorescence...." However, in our case, we saw it in one

color, while the other color scan was perfectly good, proving that the DNA spots are OK. You

go on to say that "At areas where this noise is of higher intensity, often many spots are

"inverted," that is, they are much darker than the surrounding background, almost as if the

DNA (and probe) has "peeled" away from the spot." We also saw the negative staining the in

color that failed, yet, we know that the DNA has not peeled off the slide, since the other color

worked OK. My guess is that, as you suggest, there is some problem with the probe in the

color that fails (or in your case, in both colors), but that there is nothing wrong with the slide.

In our two slides that failed, the color that failed was based on two RT reactions made from a

single polyA+ RNA prep. The negative staining could simply mean that the unincorporated

fluorochrome sticks better to alkylated glass than DNA coated surfaces. I wonder if something

like ie. phenol contamination of an RNA prep could both kill an RT reaction and also clog a

centricon, such that most of the fluorochrome in the RT reaction ends up as monomer on your

slide? We (ie. Walker) did spectral analysis using our (ie. his) hyperspectral scanner and

confirmed that the screaming high background in the color that failed was in fact spectrally the

Cy3, hence not something exogenous to the labelling experiment that happens to give a signal

in the Cy3 channel. To answer the question about metal slide-holding racks: Shandon, part

number 121 ($42), 1-800-547-7429

#131Need help to open the guide files. Can't open them with acrobat. Please help or send files in

other formats.

#134Hello, We at RoboDesign can help you with your Galil Software and MMI to run your custom

Arrayer. We can also provide you with a complete turn-key ready system. Please contact us for

a quote. Also, see our web site www.robodesign.com. Regards, Brian L. Ganz -

President/CEO RoboDesign. We are experts with the Galil DMC-1730 and servo motors.

#135There is a microarraying course planned fo the fall (october) at cold spring harbor. it is still

somewhat tentative.

#137Hi, I am writing to inquire about where I might go to find more information about the laser

used in the microarray. Thanks.

#140Let me start by congratulating all of you for your fantastic job in the area of arrays and for

maintaining the site wonderfully. Accept the wishes for a more interactive and fruitful 1999. At

NRC on DNA Fingerprinting we are working on building molecular profiles of the CROP

VARIETIES to be used subsequently for the varietal identification under TRIPs regime.

Though the work is in the infancy, we would like to adopt as much latest technique as possible

among others. May I know if any work is being done anywhere in plants (specifically in crop

plants) employing the utility of microarrays. Since I have not come across any publications (all

of which are either human, yeast or murine), could you probably be able to let me know the

labs or persons involved in such a project. Affymetrix people claim that re-use of

chips(ofcourse their make) is't possible (deprobing and reprobing in traditional terms). I am

confused and hence can not go ahead with experimentation and investment. Please reply,

Thanks again SUNIL To all of those engaged in Plant genome research: please contact me at

Archaka@yahoo.com to have a chat and hopefully to give suggestions and advice.

#144 Has anyone got experience with radioactive (P33) hybridisation on glass slides? I would like to share experiences about sensitivity.