A highly sensitive and multiplexed method for focused transcript analysis

Kari Kataja (Corresponding Author), Reetta Satokari, Mikko Arvas, Kristiina Takkinen, Hans Söderlund

Research output: Contribution to journalArticleScientificpeer-review

10 Citations (Scopus)

Abstract

We describe a novel, multiplexed method for focused transcript analysis of tens to hundreds of genes. In this method TRAC (transcript analysis with aid of affinity capture) mRNA targets, a set of amplifiable detection probes of distinct sizes and biotinylated oligo(dT) capture probe are hybridized in solution. The formed sandwich hybrids are collected on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted, optionally amplified by a PCR using a universal primer pair and detected with laser-induced fluorescence and capillary electrophoresis. The probes were designed by using a computer program developed for the purpose. The TRAC method was adapted to 96-well format by utilizing an automated magnetic particle processor. Here we demonstrate a simultaneous analysis of 18 Saccharomyces cerevisiae transcripts from two experimental conditions and show a comparison with a qPCR system. The sensitivity of the method is significantly increased by the PCR amplification of the hybridized and eluted probes. Our data demonstrate a bias-free use of at least 16 cycles of PCR amplification to increase probe signal, allowing transcript analysis from 2.5 ng of the total mRNA sample. The method is fast and simple and avoids cDNA conversion. These qualifications make it a potential, new means for routine analysis and a complementing method for microarrays and high density chips.
Original languageEnglish
Pages (from-to)102-113
Number of pages12
JournalJournal of Microbiological Methods
Volume67
Issue number1
DOIs
Publication statusPublished - 2006
MoE publication typeA1 Journal article-refereed

Keywords

  • Gene expression
  • Hybridization
  • Multiplexing
  • Transcriptional analysis

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