Development of a low-cost detection method for miRNA microarray.
MicroRNA (miRNA) microarray is a powerful tool to explore the expression profiling of miRNA. The current detection method used in miRNA microarray is mainly fluorescence based, which usually requires costly detection system such as laser confocal scanner of tens of thousands of dollars. Recently, we developed a low-cost yet sensitive detection method for miRNA microarray based on enzyme-linked assay. In this approach, the biotinylated miRNAs were captured by the corresponding oligonucleotide probes immobilized on microarray slide; and then the biotinylated miRNAs would capture streptavidin-conjugated alkaline phosphatase. A purple-black precipitation on each biotinylated miRNA spot was produced by the enzyme catalytic reaction. It could be easily detected by a charge-coupled device digital camera mounted on a microscope, which lowers the detection cost more than 100 fold compared with that of fluorescence method. Our data showed that signal intensity of the spot correlates well with the biotinylated miRNA concentration and the detection limit for miRNAs is at least 0.4 fmol and the detection dynamic range spans about 2.5 orders of magnitude, which is comparable to that of fluorescence method.
Li
Wei
W
Institute of Biochemistry and Cell Biology, The Chinese Academy of Sciences, Shanghai, China.
Zhao
Botao
B
Jin
Youxin
Y
Ruan
Kangcheng
K
D000469:Alkaline Phosphatase / Q000378:metabolism; D019921:Biotinylation; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004798:Enzymes / Q000737:chemistry; D004867:Equipment Design; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013997:Time Factors
D004798:Enzymes; D035683:MicroRNAs; D019809:Streptavidin; D000469:Alkaline Phosphatase