Guidelines for submitting RNA
Producing a high quality microarray experiment is critically dependent upon the quality of RNA samples that are produced. The technique is highly sensitive to RNA degradation and variation in quality between samples. An array experiment performed on low quality RNA rarely produces results that accurately reflect the biology of the system being examined. As such it is essential that you follow our guidelines for preparing RNA to ensure high sample quality.
- RNA Extraction. We recommend that you use Qiagen's RNeasy® kits to prepare your RNA samples, following the guidelines in the manuals that are provided with the kits. If you are not experienced with RNA isolation, please read the literature and tips found on Ambion’s website. We have produced several protocols that build upon the Qiagen manuals and may help the researcher optimize their RNA quality, available on our Protocols page. However, we strongly recommend that you also consult the kit's manual as Qiagen may have changed their protocol. If working with very small samples please try to avoid the use of carrier molecules, or consult with us for recommendations. Samples should be stored in Qiagen's Elution Buffer or water at -80°C and delivered to the BGC on dry ice.
- Quantification of RNA. RNA requirements vary upon the platform being used, but typically range from 10-500 ng. When submitting prepared RNA samples to us for expression analysis it is essential that you quantify the concentration of RNA using a spectrophotometer. We recommend the use of the Nanodrop spectrophotometer. Note: the Nanodrop is not accurate at concentrations below 10 ng/µl and for best results 1.5 µl of sample should be used when taking a reading. If you do not have access to a Nanodrop then use these guidelines for use with a regular spectrophotometer.
- RNA Quality Assessment. Microarray analysis requires RNA of very high integrity. As a standard practice all samples submitted to the BGC are analyzed for integrity using an Agilent bioanalyzer, to confirm that the ratio of 18S:28S ribosomal subunit RNA is close to the theoretical maximum of 2.5. The bioanalyzer provides a complete RNA profile with as little as 500 pg of total RNA and can quickly reveal sample degradation.
If prepared correctly, almost all RNA samples prepared from animal tissues should look like the high quality RNA trace. However, with some tissues that have a high RNase content, such as adult pancreas, a trace similar to that of the partially degraded RNA sample might be expected. Consistency in the quality of RNA samples within an experiment is critical: samples that show a high quality trace should never be compared with those that show partial degradation. At our discretion we will reject samples that we consider to be of low quality, as our experience has shown us that they will not produce reliable data.
