Wang Hui, Ach Robert A, Curry Bo
Agilent Technologies, Inc., Agilent Laboratories, Santa Clara, California 95051, USA.
RNA. 2007 Jan;13(1):151-9. doi: 10.1261/rna.234507. Epub 2006 Nov 14.
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.
我们开发了一种灵敏、准确且能进行多重检测的微小RNA（miRNA）分析方法，该方法基于一种高效标记方法和新型微阵列探针设计。这些探针可实现序列和大小区分，在大多数情况下能对密切相关的成熟miRNA进行高度特异性检测。采用简单的单管实验方案，无需分级分离或扩增，直接用Cy3或Cy5对120 ng总RNA进行标记，以产生精确且准确的测量结果，线性动态范围涵盖0.2 amol至2 fmol的输入miRNA。结果可为所研究组织的miRNA含量提供定量估计。该分析方法也适用于福尔马林固定石蜡包埋的临床样本。我们的方法能够快速设计和验证探针，以便同时对公共数据库中所有人类miRNA序列以及新报道的miRNA序列进行定量测量。
