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Project Documentation & Protocols: Maize Gene Discovery Project: Microarrays: FAQs

Contents: Index | Progress | Controls | Libraries | Arrays | Data | Protocols | Ordering | Links | FAQs

How do I get answers to my questions on getting started using microarrays?
Why do you have to buy 2 microarrays as a set?
>Can microarrays be stripped and reused or can you only use each once?
Can microarrays be custom ordered?
How do I know when microarrays will be available for a specific EST project?
Will you be posting data from experiments using the maize gene discovery project microarrays and may I share some of my results? I am having problems with microarray hybridization. Can you help?
Do I have to use spiking control? If yes, what would you recommend?


How do I get answers to my questions on getting started using microarrays?
Consult the protocol section. In addition to protocols there are instructions to microarray users with valuable tips on how to get started and where to find information. There is also a feedback page where you can send our microarray team your comments and questions.

Why do you have to buy 2 microarrays as a set?
We believe that all microarray experiments need to be replicated at least twice so we only sell them as two-slide sets.

Can microarrays be stripped and reused or can you only use each once?
Each slide can be used only once.

Can microarrays be custom ordered?
Only the EST projects that we sequence will be printed. There will be no custom orders.

How do I know when microarrays will be available for a specific EST project?
Consult the progress table where we list projects we are working on.

Will you be posting data from experiments using the maize gene discovery project microarrays and may I share some of my results?
We will be posting reproducibility data for all microarrays. In addition, we will also be posting results of experiments that project members would like to share. If you would like to share your results, please contact us through the Feedback Page and we will be happy to work with you to post your results on our site. Please send an e-mail to .

I am having problems with microarray hybridization. Can you help?
The most common question we receive concerning hybridization with our microarrays is how to reduce background. Some users find that when they use protocols that work for other microarrays, they get more background on our microarrays. We have found the following suggestions dramatically improve background levels:

  1. It is best to follow our hybridization protocol the first time because the manufacturer has optimized the protocol for the slide chemistry.
  2. It is necessary to have very clean RNA. Preparation of maize RNA can be complicated by starch and polysaccharide contaminants. For example, because of the large amount of starch in endosperm, you might need to prepare endosperm RNA using a technique that does not employ Trizol. You should carefully select a protocol that is appropriate for the tissue you are using. We recommend that as final step you use DynaBeads or some other purification step.
  3. We have found that carefully washing the labeled targets can significantly reduce background. For example, in the last step of the labeling reaction we purify the label in a Microcon-YM30 column and spin at 12000 X g for 15 min. (We have found that purification in a Microcon column is significantly better than precipitating with ethanol). We have recently begun to wash the column 2 X with 400 µl TE. Additionally, on the final wash to fully remove all unincorporated label, we often to respin 5-15 min. until liquid is no longer visible in filter. We do not allow the labeled target to completely dry. Washing labeled targets twice and fully removing liquid without over-drying are important to reducing background.
  4. Finally, we have found that not letting the microarray remain on the 65 C hot plate at hybridization is critical. In the last step of our hybridization, we place the slide on the hot plate, add hybridization mix, and quickly place the microarray into a warm moist hybridization chamber (in our case a preheated 50 ml centrifuge tube). We have found that if the slide is allowed to remain on the hot plate more than a couple of seconds, we have significantly higher background--probably because the slide dries out a little.

Do I have to use spiking control? If yes, what would you recommend?
We do recommend the use of spiking control for array hybridization. This will serve as internal control for the quality of labeling and array hybridization. However it is not absolutely necessary, because our maize microarrays contain a number of positive and negative controls to monitor the quality of the array hybridization. If the researcher decides to use spiking controls, we provide a number of human clones which have least homology to plant genes. More information can be found in the control table page.

Our previous recommendation of spiking controls pHUM1-8 is not valid anymore, because these plasmids are significantly cross hybridized with plant RNA. Our new recommendations are plasmids sp1-sp10. These plasmids will not be supplied with the array; however, they can be obtained by special request and these plasmids can be verified by PCR and sequencing. In-vitro transcription procedures are same as described in our methods section.


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