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
- Samples should either be diluted in nuclease-free water or the column's elution buffer (ie 10mM Tris). If you are using the elution buffer please describe it in the Special Instructions
Preparing RNA for microRNA Assay
The Exiqon arrays used by the VMSR for miRNA analysis start with total RNA, eliminating the troublesome fractionation step. However, you must ensure that the total RNA you submit still contains small RNAs - certain cleanup kits will eliminate them from the sample. Exiqon recommends the Qiagen miRNeasy Mini Kit, but any column guaranteed to preserve small RNA should work
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
- (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
