A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA.
We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification.
Hosoda
Kazufumi
K
Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Matsuura
Tomoaki
T
Kita
Hiroshi
H
Ichihashi
Norikazu
N
Tsukada
Koji
K
Urabe
Itaru
I
Yomo
Tetsuya
T
D001483:Base Sequence; D015342:DNA Probes; D005456:Fluorescent Dyes; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000235:genetics
D015342:DNA Probes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012313:RNA