Profiling microRNA expression using sensitive cDNA probes and filter arrays.

MicroRNAs (miRNAs) are small noncoding RNAs (approximately 22 nucleotides) that have recently emerged as important regulators of gene expression in both plants and animals. With few exceptions, however, the target genes and the expression levels of most miRNAs are unknown. Here we show that direct random-primed cDNA synthesis on either chemically synthesized small RNAs (21-22 nucleotides) or gel-purified mature miRNAs from human cells can produce specific and sensitive full-length cDNA probes. Using oligonucleotide filter arrays, we demonstrate that the internally labeled cDNA probes are sensitive for detecting differential miRNA expression between untreated and O-tetradecanoylphorbol-13-acetate (TPA)-treated HL60 cells. The present study should facilitate a high-throughput analysis of miRNA expression between samples.

[1]  F. Slack,et al.  The time of appearance of the C. elegans let-7 microRNA is transcriptionally controlled utilizing a temporal regulatory element in its promoter. , 2003, Developmental biology.

[2]  Konstantin Khrapko,et al.  A microRNA array reveals extensive regulation of microRNAs during brain development. , 2003, RNA.

[3]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[4]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[5]  T. Tuschl,et al.  Identification of Tissue-Specific MicroRNAs from Mouse , 2002, Current Biology.

[6]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[7]  D. Bartel,et al.  MicroRNAs Modulate Hematopoietic Lineage Differentiation , 2004, Science.

[8]  C. Croce,et al.  An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Russell,et al.  bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila , 2003, Cell.

[10]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[11]  Mouldy Sioud,et al.  Gene silencing by systemic delivery of synthetic siRNAs in adult mice. , 2003, Journal of molecular biology.

[12]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[13]  Qian Liu,et al.  A high-throughput method to monitor the expression of microRNA precursors. , 2004, Nucleic acids research.

[14]  M. Morley,et al.  Making and reading microarrays , 1999, Nature Genetics.

[15]  V. Kim,et al.  MicroRNA maturation: stepwise processing and subcellular localization , 2002, The EMBO journal.

[16]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.