Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes.
Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.
Pioch
Daniel
D
Pharmazeutische Biotechnologie, Institut für Pharmazie, Ernst-Moritz-Arndt Universität, Friedrich-Ludwig-Jahn-Strasse 17, 17487 Greifswald, Germany.
Jürgen
Britta
B
Evers
Stefan
S
Maurer
Karl-Heinz
KH
Hecker
Michael
M
Schweder
Thomas
T
D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors; D015374:Biosensing Techniques; D005285:Fermentation; D007218:Industrial Microbiology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger