Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.
A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.
Rautio
Jari J
JJ
VTT Biotechnology, Finland. jari.rautio@vtt.fi
Kataja
Kari
K
Satokari
Reetta
R
Penttilä
Merja
M
Söderlund
Hans
H
Saloheimo
Markku
M
D003470:Culture Media; D019075:Electrophoresis, Capillary; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D008280:Magnetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013997:Time Factors; D014158:Transcription, Genetic; D014242:Trichoderma / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism
D003470:Culture Media; D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D019809:Streptavidin