The Gene Expression Core has the following guidelines for researchers preparing RNA for microarray experiments:
- High-quality RNA is the most important determinant of successful microarray experiments. The utmost care should be taken to provide pure, nondegraded total RNA samples to the core.
- We have found that RNA prepared with Qiagen column systems works well. Care must be taken, however, to remove all residual wash buffer and ethanol from the column before the final sample elution step. We strongly recommend both spinning the column at full speed for at least two minutes, and then applying the column to a Qiagen vacuum manifold system for five minutes to evaporate the remaining ethanol before eluting the sample. Alternatively, one may spin the column for two minutes, and then move the column to a fresh empty tube and spin an additional three minutes.
- If the RNA is isolated using TRIzol, RNAzol, or a comparable procedure, then we strongly recommend using a Qiagen RNeasy cleanup kit to further purify samples.
- For larger tissue samples we recommend immediately freezing with liquid nitrogen, and then grinding to a powder with a mortar and pestle. The powder can then be placed in a TRIzol reagent or other lysis buffer. Placing chunks of tissue into lysis buffer directly often leads to RNA degradation.
- A DNase treatment is strongly recommended. It removes genomic DNA that may interfere with the experiment.
Ideal starting total RNA amounts (not mRNA) depend on the target amplification protocol requested.
- The volume of the sample should not exceed 12 ul.
- The OD 260 / 280 ratio should be at least 1.8 for pure RNA.
- Be sure to clearly label sample tubes. Include a distinct name as well as the concentration of each sample for efficient processing.
Sample Quality Testing
Before starting an experiment, the core facility performs a quality control test using the Agilent Bioanalyzer. The available assays use minimal amounts of RNA per sample. The assays separate the total RNA by size. Samples of high integrity should produce sharp, distinct 28S and 18S rRNA peaks with no evidence of degradation. Investigators should submit two microliters of sample in a well labeled 0.5 ml microcentrifuge tube. Include a printout listing the name of the samples, concentrations, and one’s contact information. Investigators may use this service without submitting samples for a microarray experiment.