Preparing Samples

Isolating RNA

In order for a microarray experiment to be successful, the starting RNA must be intact and free of contaminants. This tech note from Ambion describes several sources of contamination, purification techniques to remove them, and methods of assessing quality after extraction. It also describes the shortcomings of using rRNA ratio as the sole indicator of quality. Since the publication of this tech note, Agilent has developed the RNA Integrity Number, which assigns a quality score by examining the entire electropherogram. Two-step isolation yields much higher quality RNA and more consistent microarray results. We recommend starting with an organic solvent such as TRIzol, and then purifying a second time with a solid-state, column based kit. There are a number of high-quality column-based kits on the market; we have seen good results from RNeasy and Gentra kits. In addition, we recommend that all samples should be DNAse treated. While this is not strictly necessary for microarray work, it may be for downstream analysis and verification, and the most reliable verification will be obtained by using the same sample that was used on the microarray.

Isolating microRNA

The small size of microRNAs makes isolation and verification tricky. When isolating RNA for microRNA purification, do not use a column-based cleanup. These columns eliminate small RNAs from the sample. Once you have isolated Total RNA,

1. At the end of Trizol isolation, instead of EtOH precipitation and quantitation of total RNA, the aqueous phase is passed through a Millipore YM-100 column, and the flow-through is collected.
2. The flow-through is passed through a YM-3 column, and this time we collect what remains on the filter.