A highly sensitive and multiplexed method for focused transcript analysis

• Published in: Journal of microbiological methods 2006-10, Vol.67 (1), p.102-113
• Main Authors: Kataja, Kari, Satokari, Reetta M., Arvas, Mikko, Takkinen, Kristiina, Söderlund, Hans
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling - methods; Hybridization; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Multiplexing; Polymerase Chain Reaction - methods; RNA, Messenger - analysis; RNA, Messenger - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Transcription. Transcription factor. Splicing. Rna processing; Transcriptional analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2006.03.013

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.