Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification.
Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.
Mader
Andreas
A
Institute of Pathology, Freiburg University Medical Center, Breisacher Strasse 115a, 79106 Freiburg, Germany.
Riehle
Ulrike
U
Brandstetter
Thomas
T
Stickeler
Elmar
E
zur Hausen
Axel
A
Rühe
Jürgen
J
D001943:Breast Neoplasms / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D021161:Self-Sustained Sequence Replication / Q000379:methods*
D012313:RNA