1. How should I isolate my RNA for use in a microarray experiment?
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 a 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.
2. How do I evaluate my RNA quality?RNA quality can be evaluated by visualizing the RNA on a gel, as well as by calculating the A260/A280 ratio. On a denaturing gel (or on an ordinary agarose gel 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.
Prior to processing an array, RNA quality is checked on a bioanalyzer chip, which yields a ratio, which is the area under the peak of the 28S band over the 18S band. If the ratio of 28S to 18S is less than 1.4, it may indicate a high degree of RNA degradation, and investigators will be informed and given an option to proceed or to send new RNA.
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 < 1.8 indicate that the RNA may be contaminated with other material such as protein and salts, which may inhibit the labeling process. When this is found, the RNA should be repurified, perhaps on a column (look under RNA extraction page). RNA that is submitted with ratios < 1.8 will not yield adequate amounts of cRNA for hybridizing on a chip if the starting amount of material is set at 2 ug. Therefore, the minimum starting amount of RNA which has a ratio <1.7 will be 5 ug.
3. How much and how concentrated should the RNA be when it is sent to the facility?The RNA should be between 2.0ug - 5.0ug in 10 ul for single amplification. It should have passed the quality control procedures outlined above, and should be resuspended in RNase-free water. Currently, our methods support double amplification using as low as 10ng (in 3ul).
4. How should the RNA be sent?The RNA should be sent on dry ice via an overnight courier if being sent from outside of this institution. Locally, the RNA can be brought to the facility on wet ice. All RNA is stored at the facility at -70oC until it is used.
5. What are the costs associated with a micorarray experiment?Investigators will purchase chips on their own. The price list for arrays, please see section on pricing. For the cost for array processing will vary somewhat according to the number of chips being hybridized simultaneously. The cost will include running a bioanalyzer QC, and one positive control chip (for Affymetrix, this is the universal RNA on test3 arrays) for every run. See under Affymetrix, or contact the facility directly for more detailed pricing information.
6. How many experiments should I be doing?There is no single answer to this question, except that you probably should be doing more than one. A single array experiment is of little use, as it is impossible to sort through chip or labelling based artifacts and real biological variation in the data.
7. What labeling technique is used at the facility?The labeling techniques used within the core lab are standardized prior to being implemented for users. It should also be kept in mind that the facility is always experimenting with new technologies that become available, and so this is a moving target and can change as better technologies become available. The same can be said for the fluors used for the experiments.
9. What do I get back after an experiment is done?Users will be able to access their data as raw images and/or as processed data. The raw images (tiff files) can be used for further processing by other programs available to the researcher. The core lab will have run the standardized package for array analysis (including normalization and determination of expression fold change), and will also have done some statistical analysis (where appropriate) to generate a list of genes which are significantly changed by the treatment/condition.
10. Is there any analysis software available through the facility?The Genomics Core Laboratory has purchased several analyses software. GeneSpring and Partek Pro are available to all NIDDK researchers via floating licenses managed at CIT. GeneSpring is available to all NIDDK through our license server. Other licenses available for distribution are MAS 5.0 from the Affymetrix website. We have in addition, purchased a single copy of Spotfire and S-Plus available from our workstation.
11. How do I retrieve my data?Data can be retrieved via EDGE (Oracle Database).
12. What is the turnaround time for experiments?Generally, experiments can be completed within 2 weeks of material reaching the laboratory. This time is dependent on the number of chips that are being processed at the time.
13. Is the data archived anywhere?All data generated by the Genomics Core Laboratory is archived in EDGE.
Page last updated: May 31, 2009