RNA Preparation and Sample Quality Testing
The Microarray Core has the following guidelines for researchers who are preparing RNA for labeling:
- High quality RNA is the single most important determinant of successful microarray experiments. The utmost care should be taken to provide pure, non-degraded 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 5 minutes to evaporate 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 initially purified with 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.
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.
- Please be sure to clearly label sample tubes. Include a distinct name as well as the concentration of each sample for efficient processing.
For further information about RNA preparation, please read pages 13-16 of the Affymetrix manual [pdf].
Sample Quality Testing:
The Affymetrix Core runs each sample through quality control testing using the Agilent 2100 Bioanalyzer prior to beginning labeling procedures. The RNA 6000 Nano and Pico Assays are available which consume as little as 25-500 ng and 200-5000 pg per sample, respectively. The Bioanalyzer separates the total RNA by size — high quality samples should produce sharp, distinct 28S and 18S rRNA peaks, with a 28S to 18S ratio of 1.6-2.0. This assay tests for RNA integrity, but not for contamination. Residual wash buffer, ethanol, or other contaminants can block activity of enzymes used to prepare target.
