FAQ

1. What are microarrays?
2. How do I analyze my data?
3. How can I access the gene list for the AMF 18K Human Oligo Slide?
4. How can I access the electrospray mass spectra of the oligonucleotides used to construct the AMF 18K Human Oligo Slide?
   
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5. Are the microarrays ready to use?
6. Is it necessary to pre-hybridize the microarrays before use?
7. What is the reason for observing patches of background on my slide?
8. What does it mean when I see signals on the Arabidopsis spots when they should not light up because no RNA from Arabidopsis was added to the labelling reaction?
   
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9. How do you isolate total RNA from cells and tissues?
10. What is the minimum amount of RNA required to do a microarray experiment?
11. Are there any ways to use less than 10 ug of total RNA for labelling?
12. Are un-hybridized arrays kept longer than 2 months still useable?
13. How can I spin my slides dry if I don't have a plate-holder adapter for my centrifuge?
14. How long will the fluorescent signal remain on the slide?
15. How can I reduce background?
16. Can I re-wash my slides?
17. Can I re-use my slides?

1. What are microarrays?

• A microarray consist of an orderly arrangement of molecules (DNA fragments, antibodies, aptamers ..) or cells positioned on a specific location on a dimensionally-rigid array (usually glass or silicon slide). In DNA microarrays each DNA fragment (cDNA or oligonucleotide) represents one gene of an organism.
• In spotted DNA microarrays each DNA fragment is spotted, using microcontact or inkjet methods, to an assigned location. Highly accurate robotic spotters can place over 40,000 spots on one slide.
• Single-stranded DNA fragments are strongly attached to the slide (through covalent bonds or electrostatic attraction). Fluorescently labelled DNA or RNA will hybridize to a complementary nucleic acid strand at a given spot, i.e. gene fragment, on the array.
• The intensity of the fluorescence signal will be proportional to the amount of the labelled genetic material hybridized to the spotted gene fragment, i.e. gene expression or DNA copy number.

2. How do I analyze my data?

The analysis of microarray data depends on the questions asked in the experiment as well as the experimental design and the quantity of microarray data available. Numerous analysis options and software are available (both commercial and open-source). They would help you do some of the work but meaningful application of these tools can only be achieved with complete understanding of the principles defining them.

3. How can I access the gene list for the AMF 18K Human Oligo Slide?

We will provide gal file which includes UniGene ID's for all genes on the slide.

4. How can I access the electrospray mass spectra of the oligonucleotides used to construct the AMF 18K Human Oligo Slide?

Upon request, we can provide quality index information for any of oligonucleotides used on the slide.

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5. Are the microarrays ready to use?

Our oligonucleotide microarrays are presently spotted on epoxide-coated glass slides. It is highly recommended to block the slide surface prior to hybridization as it reduces the background signal significantly.

6. Is it necessary to pre-hybridize the microarrays before use?

No, it is not necessary.

7 . What is the reason for observing patches of background on my slide?

There are several causes of overall background found on the slide:

• Incomplete purification of labelling reaction - unincorporated Cy-dyes will stick to the arrays and give rise to flurorescent background upon scanning.
• Drying of hybridization buffer during overnight incubation - causing the coverslip to stick to the arrays and making it difficult to slide off in the washing solution.

Remedy:

• Follow the manufacturers protocol of the labelling procedure used to remove all un-incorporated dyes before hybridization.
• Ensure the hybridization chamber is tightly sealed.
• When background is observed during pre-scanning of the spotted slide, wash it in either higher temperature or lower salt concentration (0.1x SSC with 0.1%SDS) to increase stringency of washes. This will reduce background on slides.

8. What does it mean when I see signals on the Arabidopsis spots when they should not light up because no RNA from Arabidopsis was added to the labelling reaction?

This is caused by non-specific binding of the labelled probe on the Arabidposis spots. Although non-specific binding can be a problem with any type of DNA microarray it is much lower in oligonucleotide arrays.

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9. How do you isolate total RNA from cells and tissues?

There are many excellent kits available for the isolation of total RNA from cells and tissues. We currently use Qiagen's RNeasy kit for total RNA isolation from cells and Trizol® reagent from Gibco BRL for total RNA isolation from tissue samples.

 

10. What is the minimum amount of RNA required to do a microarray experiment?

Generally, we use total RNA for our experiments. With our direct labelling protocol we recommend using 10 µg of total RNA. Our indirect protocol also calls for 10 µg of total RNA but we have found that we can reduce this requirement to 5 µg if necessary without much loss of signal. We do not recommend using less than 10 µg for our direct protocol. Certain tissues also seem to be somewhat problematic and may require more RNA.

11. Are there any ways to use less than 10 ug of total RNA for labelling?

There are amplification labelling methods that can be used to reduce the amount of starting material required.

12. Are un-hybridized arrays kept longer than 2 months still useable?

Although un-hybridized arrays can be stored for many months at room temperature in a dry environment, we don't recommend using arrays after 8 weeks from the day of printing.

13. How can I spin my slides dry if I don't have a plate-holder adapter for my centrifuge?

There are two alternatives for drying slides. One alternative is to use compressed air to "blow-dry" the slides. We advise users to place a 0.2 µm filter on the end of the tubing to prevent particles and oil being deposited on the slides during the drying process. The other method of drying slides is to use a 50 mL Falcon tube with a few small Kim-Wipes tightly stuffed into the bottom. Place your slide inside the tube using forceps to handle the slide at the barcode-end. Spin in a 50 mL tube-sized bucket at 500 rpm for 5 minutes.

14. How long will the fluorescent signal remain on the slide?

While it is ideal to scan your slides immediately after washing/drying (especially in summer or high ground-level ozone areas), you can scan your slides 2-3 days after washing. Be sure to store your hybridized slides in the dark, and preferably in a cool and dry place. Coating the slides with a product such as Genisphere's DyeSaver may reduce dye degradation.

15. How can I reduce background?

There are several factors that lead to preventable background. First, be sure that you have DIG Easy Hyb (or whatever hyb solution you are using) at the bottom of your hyb chamber as this prevents the drying of the hyb solution and labelled-cDNA to the array. Be sure that your washes are stringent enough. Be sure to rinse your slides well after washing in SDS (or other detergents). Detergents can form micelles that can trap un-incorporated fluorophore.

16. Can I re-wash my slides?

Slides can be re-washed and sometimes "swirly" background from incomplete washing/rinsing can be removed. However, re-washing can also remove some of the signal. We strongly recommend that you always save your image before re-washing the slide (in case all signal is removed). The length of re-wash, temperature and washing buffer used all vary between users. As a starting point, we would suggest trying a 5 minute re-wash at 50 0 C using 1X SSC/0.1% SDS (usual wash buffer) if necessary.

17. Can I re-use my slides?

No, slides are not re-usable; they can only be used for one hybridization.