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Gene Microarray FAQ

1. How should I isolate my RNA for use in a microarray experiment?
2. How do I evaluate my RNA quality?
3. How concentrated should the RNA be when it is sent to the facility?
4. How should the RNA be sent to the facility?
5. What if I need a chip that is not listed as in stock at the facility?
6. What are the costs associated with a microarray experiment?
7. How many experiments should I be doing?
8. What technique is used at the facility to label the cDNA?
9. What do I get after an experiment is done?
10. Is there any analysis software available through the facility?
11. Are there options for help with data analysis?
12. How do I retrieve my data?
13. What is the turnaround time for experiments?
14. Are the data archived anywhere?
15. Are there any good sources of information on microarrays that I can access?

#1 How should I isolate my RNA for use in a microarray experiment?
Answer: The main objective is to generate RNA that is of high quality and sufficiently concentrated for use in a microarray experiment. Many researchers have found that isolation with Trizol reagent, followed by column purification using the RNEasy Total RNA Isolation Kit (Qiagen), generates RNA of sufficient quality for these experiments. However, each type of starting material may generate RNA of different quality and no one technique is likely to work for all samples. The microarray facility now offers RNA extraction services from a variety of sources. These include whole blood (collected in PAX, EDTA or Na citrate collection tubes), white cell pellets, frozen tissue, cell lines, and laser-capture microdissected samples from either frozen sections or formalin-fixed paraffin-embedded (FFPE) tissue. Protocols for these extractions can be viewed under the protocols link. Fees for these services are listed on the fee schedule.

Frozen tissues in RNAlater-ICE (Ambion #7030) are also processed as described above.

#2 How do I evaluate my RNA quality?
Answer: RNA quality can be evaluated by visualizing the RNA on a gel, and also by calculating the A260/A280 ratio. On a denaturing gel (or on non-denaturing agarose in denaturing buffer) the RNA should appear as two bright discrete bands that represent the 28S and 18S ribosomal species. The 28S band should be brighter than the 18S band. Tailing of these major bands down the gel, or a background smear behind these bands that gets heavier at lower molecular weights can indicate degradation of the RNA. In these cases, it is best to isolate fresh RNA, as degraded RNA will produce high background and low signal intensities on a microarray. The presence of very sharp bands higher than the 28S ribosomal band can indicate the presence of excess DNA in the sample, which can be removed by treatment with RNase-free DNase. The spectrophotometric ratio will also give an indication of the purity of the RNA, and should be as close to 2.0 as possible. Generally, ratios less than 1.7 indicate that the RNA may be contaminated with other material, and should be re-purified, perhaps on a column.

Another method, available in our facility, is the use of an Agilent 2100 Bioanalyzer. This instrument uses rapid capillary-electrophoresis to analyze DNA, RNA, or protein samples. It generates both an electropherogram (line graph) and a pseudo-gel image as visual aids to your analysis. The numerical data generated are dependent on the type of assay selected. One major advantage in the analysis of total RNA is that an RNA Integrity Number (RIN) is generated which correlates to the quality of the sample.

All samples extracted in our facility are assessed using the Bioanalyzer.

#3 How concentrated should the RNA be when it is sent to the facility?
Answer: The RNA should be resuspended to at least 1.0 µg/µL in RNase-free water. It should have passed the quality control procedures outlined above or, alternatively, you can ask that we assess the quality using the Agilent 2100 Bioanalyzer. The cost for this service is outlined in the fee schedule.

Amplification can be done in cases of very low amounts of RNA. Amplifications for two-colour microarrays require a minimum of 50 ng of RNA at a concentration of at least 2.5 ng/µL. Those for one-colour microarrays require at least 200 ng of RNA at a concentration of at least 10 ng/µL. There is an additional charge for amplifications. Please contact us regarding the price.

#4 How should the RNA be sent to the facility?
Answer: The RNA should be sent on dry ice via overnight courier if being sent from outside of this institution. From within the Queen's and KGH area, the RNA can be brought to the facility on ice. All RNA is stored at the facility at -80°C until it is used and, following experimentation, the remainder is stored long-term at -80°C.

#5 What if I need a chip that is not listed as in stock at the facility?
Answer: The facility will assist in tracking down chips that may be of interest to individual researchers and the chips can be purchased by either the facility or the researcher. If the facility purchases the chips, the cost is passed on to the user, along with any applicable shipping charges.

#6 What are the costs associated with a microarray experiment?
Answer: The cost will vary somewhat according to the price of the chip being used. The user is referred to the pricing section of this web page or can contact the facility directly for more detailed pricing information.

#7 How many experiments should I be doing?
Answer: There is no single answer to this question, except that you should be doing more than one. A single array experiment is of little use, as it is impossible to sort through chip or labeling-based artifact and real biological variation in the data. The generally recommended minimum is triplicate experiments. Both technical and biological replicates should be considered. Contact us to discuss your particular experimental design.

#8 What technique is used at the facility to label the cDNA?
Answer: Several labeling strategies are currently in use at the facility. We most commonly use an Agilent Technologies two-colour direct-labeling protocol which requires a starting amount of 10-20 µg of total RNA per channel. Please contact us to discuss which protocol best suits your needs.

Agilent One-Colour and Two-Colour labeling protocols can be found at www.agilent.com . (Select "Manuals & Guides", then "Life Sciences and Chemical Analysis Solutions".)

#9 What do I get after an experiment is done?
Answer: A headache.

What do I really get?

You will get the scanned images of the chips for both fluors as TIF images. The images can be used as input into your own quantitation program, should you have access to one that you prefer to use. You will also get a spreadsheet that consists of a number of pages that include the raw data generated from quantitation of the scanned images. You will also receive quality control parameters for each experiment.

#10 Is there any analysis software available through the facility?
Answer: Contact us to discuss your individual needs.

#11 Are there options for help with data analysis?
Answer: Contact us to discuss your individual needs.

#12 How do I retrieve my data?
Answer: Data can be retrieved in a couple of ways. An FTP site is set up on the facility computer with a private account for your data. You will be sent the address of the relevant computer, your FTP site username and password. You can then access the site to download the data to your own computer. Alternatively, the data can be sent on a CD. There is an additional charge for this.

#13 What is the turnaround time for experiments?
Answer: Generally, experiments can be completed within 2 weeks of material reaching the laboratory, provided we have been given sufficient lead time to obtain the required chips. We do not tend to hold large numbers of unused chips and prefer to order fresh chips for your experiments. Please note that it can take up to 2 weeks to obtain chips.

#14 Are the data archived anywhere?
Answer: All data generated by the microarray facility are archived on CDs for long term storage. Should you not want your data archived in this manner, simply inform the facility that you wish all of your data to be deleted following download of your files.

#15 Are there any good sources of information on microarrays that I can access?
Answer: There are many sources of information about microarrays available on the web. A few sites are listed below. The information in these sites changes constantly, so it is always a good idea to check periodically to see what is new.