Working with Coriell

working_with_coriellWhen you are ready to start a project, Coriell’s Genotyping and Microarray Center can design a custom service for your research needs.

Following is some basic information to begin our collaboration:

  • For expression profiling, isolate either total RNA (most common) or polyA-selected RNA.
  • For genotyping, isolate genomic DNA. See our recommendations for DNA/RNA isolation below.
  • For small sample sets, it generally takes about two weeks for us to process your samples, hybridize them to the GeneChips, collect the data, and present you with electronic and/or hard copies of the results.
  • If you are unfamiliar with microarray analysis, we can order GeneChips for you.

Getting started with Affymetrix Expression Profiling GeneChips is easy:

  • Set-up an appointment with members of the Microarray Center to discuss your experiment. This is optional, but highly recommended.
  • Provide us with your total RNA samples. Standard processing is available starting with as little as 500 pg of total RNA.
  • Wait 1-2 weeks for us to process your samples.
  • Download your data files from our secure server for analysis.

For more information, please contact: Norman Gerry, PhD, Director, Coriell Molecular Biology.

 

genotyping-microarray-isolating-dna-rnaRNA 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 have not isolated RNA from your system before, 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.

 

RNA Purity

Always run an agarose gel of your RNA to assess the quality prior to sending us your sample. If you use a non-column based purification reagent such as TRIzol, we recommend you precipitate your RNA a second time from ethanol, or pass it through an RNeasy column or similar device. This insures removal of all organic contaminants.

A260 readings and 260/280 ratios are not enough to ensure high purity RNA. You must take a full UV spectrum. Here we illustrate 3 sample spectra which demonstrate 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.

genotyping-microarray-isolating-dna-rna-2UV 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 and Tissue
For the initial purification of RNA from mammalian tissue, Affymetrix recommends using TRIzol Reagent. For subsequent sample “clean-up” Affymetrix recommends using QIAGEN’s RNeasy Total RNA Isolation kit.

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).

Two DNA isolation procedure recommended by Affymetrix are SDS/ProK digestion, phenol-chloroform extraction, Microcon or Centricon (Millipore) ultrapurification and concentration; and also, QIAGEN’s 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.