Rational probe optimization and enhanced detection strategy for microRNAs using microarrays

Loyal A. Goff, Maocheng Yang, Jessica Bowers, Robert C. Getts, Richard W. Padgett, Ronald P. Hart

Research output: Contribution to journalArticle

Abstract

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Original languageEnglish (US)
Pages (from-to)93-100
Number of pages8
JournalRNA Biology
Volume2
Issue number3
DOIs
StatePublished - Jan 1 2005
Externally publishedYes

Keywords

  • Oligonucleotide arrays
  • Post-transcriptional regulation
  • Stem cells

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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