Improved representation of gene markers on microarray by PCR-Select subtracted cDNA targets

E. Yakubov, P. Dinerman, F. Kuperstein, S. Saban, E. Yavin

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Despite the success and popularity of microarrays as a high-throughput technology for gene-expression studies, its sensitivity is as yet fairly limited. We have successfully combined the use of PCR-Select cDNA subtraction and Affymetrix GeneChips (AGC) to identify differentially expressed gene markers. Total RNA (totRNA) from combined hippocampus and cerebellum tissues of 2-week-old rat pups maintained for 5 weeks on an n-3 fatty acid (FA) deficient diet supplied to dams was isolated, SMART-amplified, and used for PCR-Select subtraction versus an adequately fed control litter preparation. Subtracted and amplified ds-cDNA end products were fragmented, terminally labeled with biotin-ddUTP and hybridized with RN-U34A AGC. At least 10-fold more potential gene markers with log2(T/D) ≥ 1.4 were found versus the traditional AGC technology when the same chip was tested using nonsubtracted targets. Of this set of markers, 30% were robustly validated by real-time relative RT-PCR (rtrRT-PCR) and grouped as "confirmed" markers while the remaining were ascribed as "latent" markers. An improved and universal protocol to provide a rapid assessment for gene profiling in biological specimens is indicated.

Original languageEnglish (US)
Pages (from-to)110-118
Number of pages9
JournalMolecular Brain Research
Volume137
Issue number1-2
DOIs
StatePublished - Jun 13 2005

Keywords

  • Affymetrix GeneChips
  • Gene markers
  • n-3 fatty acids
  • Suppression subtractive hybridization

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

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