Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.
BACKGROUND
Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions.


RESULTS
Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt.


CONCLUSIONS
We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.
Scheler
Ott
O
Department of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. ott.scheler@ut.ee
Glynn
Barry
B
Parkel
Sven
S
Palta
Priit
P
Toome
Kadri
K
Kaplinski
Lauris
L
Remm
Maido
M
Maher
Majella
M
Kurg
Ants
A
D005456:Fluorescent Dyes / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000737:chemistry*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000378:metabolism; D014544:Uridine Triphosphate / Q000737:chemistry
D005456:Fluorescent Dyes; D012329:RNA, Bacterial; C096222:tmRNA; D014544:Uridine Triphosphate