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Replacing cRNA targets with cDNA reduces microarray cross-hybridization

Nature Biotechnology volume 24, pages 1071–1073 (2006)Cite this article

To the editor:

Gene-expression microarrays are designed to measure relative concentrations of transcripts through the specific hybridization of an immobilized DNA probe to its complementary target. This technology is viable to the extent that a single, rather permissive hybridization condition allows most probes to bind specifically to their targets. Despite efforts to maximize stringency, a significant hybridization signal can still be detected on various oligonucleotide-based platforms, even when there are a few mismatches between probe and target1,2,3. Furthermore, several groups have detected widespread cross-hybridization in microarray measurements4,5, and on the order of 10% of the probes on a common oligonucleotide array platform were predicted to be susceptible to cross-hybridization5. Efforts to optimize probe length found that longer probes enjoy stronger signal intensity but also suffer from increased propensity toward cross-hybridization6. Therefore, nonspecific binding remains a significant source of measurement error and may be the reason why quantitative reverse transcription (qRT)-PCR fails to confirm about 10–20% of difference calls made by microarray analysis (reviewed in ref. 7). Here, we report that a high-level of promiscuity in DNA-RNA hybridization underlies widespread cross-hybridization in microarrays. This cross-hybridization can be reduced using cDNA targets in place of cRNA.

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Figure 1: Comparison of microarray data from Jurkat-cell RNA with and without spiked transcripts.

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Authors and Affiliations

  1. Children's Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology (CHIP@HST), Harvard Medical School, Boston, 02115, Massachusetts, USA

    Aron C Eklund & Zoltan Szallasi

  2. Center for Neurologic Diseases, Brigham and Women's Hospital, 65 Landsdowne St., Cambridge, 02139, Massachusetts, USA

    Aron C Eklund

  3. NuGEN Technologies, Inc., 821 Industrial Rd, Unit A, San Carlos, 94070, California, USA

    Leah R Turner, Pengchin Chen, Gianfranco deFeo & Anne R Kopf-Sill

  4. Department of Physics, University of Massachusetts Boston, Boston, 02125, Massachusetts, USA

    Roderick V Jensen

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  1. Aron C Eklund
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Eklund, A., Turner, L., Chen, P. et al. Replacing cRNA targets with cDNA reduces microarray cross-hybridization. Nat Biotechnol 24, 1071–1073 (2006). https://doi.org/10.1038/nbt0906-1071

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