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Timothy G. Myers, Ph.D.
Bldg. 50, Rm. 5509
50 South Drive, MSC 8005
Bethesda, MD 20892-8005
Phone: 301-496-0502
Fax: 301-496-0449

Research Technologies Branch

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Oligonucleotide Microarray Platform

Microarray Unit staff will conduct all probes on the current MRF microarrays that are composed of long (60- to 70-mer) oligonucleotides. We consider this format to be the best balance between sensitivity, sequence specificity, and accuracy.

The oligonucleotide design process is an important aspect of identifying the best sequences to serve as sensors of gene expression. In conjunction with commercial providers, oligonucleotide design specifications were created to help optimize the sequence selection for microarray use.

The specifications include:

  • Length: 70 bases
  • Modification: 5'-C6 amino modifier
  • Oligo synthesis efficiency: <99%
  • Reduced propensity to form secondary structure
  • Minimized cross-hybridization potential to non-target mouse or human genes
  • Consistent theoretical melting temperature with all other oligonucleotides
  • Bias to the 3' end of the gene for enhanced cDNA labeling
  • Targeted sequence region of high sequence fidelity and accuracy
  • Positive and negative controls provided
  • Oligonucleotides purified for enriched full-length product and desalted

Version 2 Microarray Oligonucleotide Sets

The standard mouse and human microarrays in current production are based on the second version of oligonucleotide libraries. The version 2 oligonucleotides were completely redesigned based on RefSeq and UniGene databases (UniGene builds Hs147 for human and Hm102 for mouse). For details of the genome set design parameters, see the Qiagen Array-Ready Oligo Sets* and for oligo reports and the estimated potential for cross-hybridization, see the Qiagen Oligo Microarray Database.* Because different (and presumably better) oligonucleotide selection criteria were used to design version 2 probes, very few of the sequences are identical to those found in the version 1 sets. Investigators can request microarrays printed with version 1 libraries while supplies last.

Control Probes on Every Microarray

Standard microarrays come with several types of control probes that can help investigators better understand the performance of each experiment. Prelabeled controls are located in the top left corner of every block on a standard microarray. These controls help the user correctly align the grids onto the arrays during data extraction using GenePix Pro 4.0 software. Negative controls are randomized sequence probes used to estimate the level of non-specific binding to the array. The MRF will be printing Ambion's ArrayControl probes and Stratagene's SpotReport Alien Oligo Array Validation System probes on every array. Investigators have the option of purchasing the sets of different artificial RNA standards to spike into the labeling reaction as external controls. This can be used to assess the sensitivity and dynamic range of each experiment.

Probe Sequence Information

The NIAID Division of Intramural Research (DIR) has signed a nondisclosure agreement with Qiagen to allow access to the complete lists of sequences for every probe on both version 1 and version 2 microarrays. This allows investigators to perform any type of additional data mining or validation studies. Although the agreement is generous in what it enables investigators to do from a scientific perspective, it does prohibit the bulk release of these sequences into the public domain. Therefore, anyone who is interested in receiving the complete file of probe sequences should contact Timothy G. Myers, Ph.D., for more information about the agreement.

MRF Adopts Epoxy-Coated Slides

MRF now prints standard arrays on epoxy-coated slides instead of the more conventional poly-lysine coating. Investigators who are using slides from any of the more recent microarrays should notice improved slide performance due to the new epoxy-coated slides. The commercially available epoxy slides show a more consistent background and better signal-to-noise ratios. This, in turn, produces more consistent measurements of gene expression ratios across the dynamic range of the experiment so that the detection of significant differences between RNA samples is more sensitive.

*Note: Some of the links on this page connect to information sources outside of NIAID and are provided as a convenience for World Wide Web users. Please see the NIAID disclaimer.

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Last Updated September 30, 2008