Abstract
Moving of biological research to postgenomic era with a growing number of
organisms including filamentous fungi has increased the interest in functional
genomics, and the need for fast and reliable transcriptional profiling methods is
thus growing. We have developed a rapid method for transcriptional profiling of
microbial cultivations based on a novel technique called TRAC "transcriptional
profiling with the aid of affinity capture". This method allows fast gene
expression analysis for sets of mRNAs by solution hybridisation with a pool of
target-specific oligonucleotide probes of distinct sizes that are identified and
quantified by capillary electrophoresis. The assay procedure has been semiautomated for simultaneous treatment of 96 samples using magnetic bead
particle processor. To further enhance the robustness of the method it was set
up to work with crude cell lysates. TRAC has been shown to produce results
highly consistent with mRNA quantification by Northern hybridisation.
Computational methods have been applied for design of target-specific
oligonucleotide probes and to assign them into minimal number of pools. The
whole assay procedure can be performed in three hours, implying its usefulness
in bioprocess monitoring and control. The developed TRAC method application has been used for monitoring the levels of a set of mRNAs in the filamentous fungus Trichoderma reesei in fermentation conditions. Chosen gene markers for bioprocess monitoring are involved in various cellular pathways including unfolded protein response, protection against various stress conditions, oxygen and nutrient limitation responses, protein synthesis and growth. Data collected from different types of fermentations, batch, fed-batch and continuous cultures, shows the potential of the method for use in optimisation of production processes and provides novel information about regulation of various genes during different phases cultivations. We have also used TRAC successfully for assessment of steady states in chemostat cultures.
organisms including filamentous fungi has increased the interest in functional
genomics, and the need for fast and reliable transcriptional profiling methods is
thus growing. We have developed a rapid method for transcriptional profiling of
microbial cultivations based on a novel technique called TRAC "transcriptional
profiling with the aid of affinity capture". This method allows fast gene
expression analysis for sets of mRNAs by solution hybridisation with a pool of
target-specific oligonucleotide probes of distinct sizes that are identified and
quantified by capillary electrophoresis. The assay procedure has been semiautomated for simultaneous treatment of 96 samples using magnetic bead
particle processor. To further enhance the robustness of the method it was set
up to work with crude cell lysates. TRAC has been shown to produce results
highly consistent with mRNA quantification by Northern hybridisation.
Computational methods have been applied for design of target-specific
oligonucleotide probes and to assign them into minimal number of pools. The
whole assay procedure can be performed in three hours, implying its usefulness
in bioprocess monitoring and control. The developed TRAC method application has been used for monitoring the levels of a set of mRNAs in the filamentous fungus Trichoderma reesei in fermentation conditions. Chosen gene markers for bioprocess monitoring are involved in various cellular pathways including unfolded protein response, protection against various stress conditions, oxygen and nutrient limitation responses, protein synthesis and growth. Data collected from different types of fermentations, batch, fed-batch and continuous cultures, shows the potential of the method for use in optimisation of production processes and provides novel information about regulation of various genes during different phases cultivations. We have also used TRAC successfully for assessment of steady states in chemostat cultures.
| Original language | English |
|---|---|
| Title of host publication | 8th European Conference on Fungal Genetics |
| Editors | Irina S. Druzhinina, Alexey G. Kopchisnkiy, Christian P. Kubicek |
| Publisher | Vienna University of Technology |
| Chapter | VII Cell-Factories and Biotechnology |
| Pages | 251 |
| Publication status | Published - 2006 |
| Event | 8th European Conference on Fungal Genetics - Vienna, Austria Duration: 8 Apr 2006 → 11 Apr 2006 |
Conference
| Conference | 8th European Conference on Fungal Genetics |
|---|---|
| Country/Territory | Austria |
| City | Vienna |
| Period | 8/04/06 → 11/04/06 |