Direct and sensitive miRNA profiling from low-input total RNA.
We have developed a sensitive, accurate, and multiplexed microRNA (miRNA) profiling assay that is based on a highly efficient labeling method and novel microarray probe design. The probes provide both sequence and size discrimination, yielding in most cases highly specific detection of closely related mature miRNAs. Using a simple, single-vial experimental protocol, 120 ng of total RNA is directly labeled using Cy3 or Cy5, without fractionation or amplification, to produce precise and accurate measurements that span a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. The results can provide quantitative estimates of the miRNA content for the tissues studied. The assay is also suitable for use with formalin-fixed paraffin-embedded clinical samples. Our method allows rapid design and validation of probes for simultaneous quantitative measurements of all human miRNA sequences in the public databases and to new miRNA sequences as they are reported.
Wang
Hui
H
Agilent Technologies, Inc., Agilent Laboratories, Santa Clara, California 95051, USA. hui_wang@agilent.com
Ach
Robert A
RA
Curry
Bo
B
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000032:analysis*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015347:RNA Probes / Q000737:chemistry; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
D002232:Carbocyanines; D035683:MicroRNAs; D015347:RNA Probes; C089187:cyanine dye 3; C085321:cyanine dye 5