A high-throughput microRNA expression profiling system.

As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings.

[1]  T. Tuschl,et al.  Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex , 2008, Nature.

[2]  Shangqin Guo,et al.  MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. , 2008, Developmental cell.

[3]  Todd R. Golub,et al.  MicroRNA miR-125a controls hematopoietic stem cell number , 2010, Proceedings of the National Academy of Sciences.

[4]  C. Xiao,et al.  An in vivo functional screen uncovers miR-150-mediated regulation of hematopoietic injury response. , 2012, Cell reports.

[5]  Thomas Tuschl,et al.  miRNAs in human cancer , 2011, The Journal of pathology.

[6]  Thomas Tuschl,et al.  Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. , 2008, Methods.

[7]  C. Croce,et al.  MicroRNAs in normal and malignant hematopoiesis , 2008, Current opinion in hematology.

[8]  T. Golub,et al.  MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia , 2007, Proceedings of the National Academy of Sciences.

[9]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[10]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[11]  G. Church,et al.  Efficient microRNA capture and bar-coding via enzymatic oligonucleotide adenylation , 2008, Nature Methods.

[12]  H. Zhang,et al.  MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer. , 2009, Genes & development.

[13]  Jun Lu,et al.  Complex oncogene dependence in microRNA-125a–induced myeloproliferative neoplasms , 2012, Proceedings of the National Academy of Sciences.

[14]  T. Golub,et al.  Distinct microRNA expression profiles in acute myeloid leukemia with common translocations , 2008, Proceedings of the National Academy of Sciences.

[15]  S. Fulmer-Smentek,et al.  Agilent microRNA microarray profiling system. , 2012, Methods in molecular biology.

[16]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.