Quantitation of microRNAs using a modified Invader assay.
The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.
Allawi
Hatim T
HT
Third Wave Technologies, Inc., 502 South Rosa Rd., Madison, WI 53719, USA. hallawi@twt.com
Dahlberg
James E
JE
Olson
Sarah
S
Lund
Elsebet
E
Olson
Marilyn
M
Ma
Wu-Po
WP
Takova
Tsetska
T
Neri
Bruce P
BP
Lyamichev
Victor I
VI
D001483:Base Sequence; D002460:Cell Line; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry
D035683:MicroRNAs; D009838:Oligodeoxyribonucleotides