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.
Our experience in the VMSR has shown that Ambion is correct, and a 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, the VMSR recommends the following:
- Affymetrix samples should not be treated with Β-mercaptoethanol. Affymetrix has informed us that BME can cause high background on GeneChips
- 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 DNA
Starting material for a genotyping microarray must meet the following requirements:
- DNA must be free of PCR and enzyme inhibitors (such as heme, high concentrations of chelating agents, or high concentrations of salts)
- DNA must not be contaminated by genomic DNA from other humans or other organisms
- DNA must not be highly degraded
- (DNA Mapping Only) DNA must be double-stranded
- (500K DNA Mapping Only) DNA may be pre-amplified with the QIAGEN Repli-G Kit. Any amplification performed with other kits or on samples destined for other array types has not been tested by Affymetrix. Such samples would be run at the PI's risk.
The following extraction/purification methods have been tested by Affymetrix:
- SDS/ProK digestion, phenol-chloroform extraction, Microcon® or Centricon® (Millipore) ultrapurification and concentration
- QIAGEN; QIAmp® DNA Blood Maxi Kit
- (Targeted Genotyping Only) Gentra PUREGENE
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, you have two options:
- Submit the sample to us for microRNA isolation using a fractionator
- OR follow this protocol (provided to us by Combimatrix):
- 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.
- The flow-through is passed through a YM-3 column, and this time we collect what remains on the filter.
- The collected material is EtOH precipitated [Note that Ambion recommends using a 4x volume of ethanol].
