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Nucleic Acid Isolation Protocols |
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RNA Isolation
Many different RNA isolation protocols are compatible with the Affymetrix GeneChip system. The only requirement is that you are able to isolate high-quality intact RNA that does not contain significant amounts of contaminating DNA.
If you currently have a preferred method for isolating RNA, continue to use it.
If you haven't isolated RNA from your system before and your organism is not on the following list, we can help you identify established protocols that have been used for isolating RNA for Northern blotting or RT-PCR from your system. These types of protocols will also yield "microarray-grade" RNA.
Always run an agarose gel of your RNA to assess the quality.
NOTE: If you use a non column based purification reagent such as TRIzol we recommend you precipitate your RNA a second time from ethanol or you pass it through an RNeasy column or similar device. This insures removal of all organic contaminants (See spectra below).
RNA Purity
A260 readings and 260/280 ratios are not enough to ensure high purity RNA. You must take a full UV spectrum. Below are 3 example spectra which all have good 260/280 ratios, but have very different purities. You can also use the 260/230 ratio to help determine the amount of organic contamination in your RNA. It is a much more variable number than the 260/280 ratio, but generally, ratios above 1 indicate good purity.
UV Spectra of RNA Samples of Varying Purity
All three samples appeared clean when just taking the A260 and 260/280 ratios. It only becomes clear that they have very different purities when a full UV spectrum is taken.
Mammalian Cell Culture
Affymetrix recommends using QIAGEN's RNeasy Total RNA Isolation kit. Link
to RNeasy product info.
Mammalian Tissue
Affymetrix recommends using TRIzol Reagent for the initial purification
of RNA from mammalian tissue. This is followed by a subsequent sample "clean-up" using QIAGEN's RNeasy Total RNA Isolation kit (see above). Link to TRIzol product info.
DNA isolation
Just as for RNA, many different DNA isolation protocols are compatible with the Affymetrix platform. The general requirements for genomic DNA quality are:
- DNA must be double-stranded
- DNA must be free of PCR inhibitors (i.e. high concentration of heme, chelating agents, or salts)
- DNA must not be contaminated with other human genomic DNA sources, or with genomic DNA from other organisms
- DNA must not be highly degraded (on a 1-2% agarose gel it should run as a major band at 10-20 KB)
Recommended Protocols
Two DNA isolation procedure recommended by Affymetrix are:
1. SDS/ProK digestion, phenol-chloroform extraction, Microcon or Centricon (Millipore) ultrapurification and concentration.
2. QIAGEN; QIAamp DNA Blood Maxi Kit.
For submission to the Coriell Genotyping and Microarray center, genomic DNA must be quantitated by PicoGreen (Molecular Probes) and provided at a concentration of 50 ng/µL in reduced EDTA TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA, pH 8.0). Failure to follow these requirements can lead to sample failures which will be the responsibility of the investigator.
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