Detection of a thousand copies of miRNA without enrichment or modification.
We report direct quantitative analysis of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to determine levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7). A 30 pL volume of the cell lysate with approximately a material content of a single cell was sampled for the analysis. MiRNA 21, which is up-regulated in breast cancer, was detected at a level of approximately 12 thousand copies per cells. MiRNAs 125b and 145, which are down-regulated in breast cancer, were below the 1000-copy detection limit. This sensitive method may facilitate the analysis of miRNA in fine-needle-biopsy samples and even in single cells without enrichment or modification of miRNA. Advantageously, the instrumental setup developed here can be reproduced by others as it requires no sophisticated custom-made parts.
Bryan J
BJ
Dodgson
Amir
A
Mazouchi
David W
DW
Wegman
Claudiu C
CC
Gradinaru
Sergey N
SN
Krylov
D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004867:Equipment Design; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
C531888:MIRN125 microRNA, human; C541010:MIRN145 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs