Real-time quantification of microRNAs by stem–loop RT–PCR

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

[1]  M. Barton,et al.  MicroRNA binding sites in Arabidopsis class III HD-ZIP mRNAs are required for methylation of the template chromosome. , 2004, Developmental cell.

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

[3]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[4]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[5]  Sarah Olson,et al.  Quantitation of microRNAs using a modified Invader assay. , 2004, RNA.

[6]  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.

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

[8]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

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

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

[11]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[12]  Gary Ruvkun,et al.  Identification of many microRNAs that copurify with polyribosomes in mammalian neurons , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Oliver Hobert,et al.  A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans , 2003, Nature.

[14]  Michael Z Michael,et al.  Reduced accumulation of specific microRNAs in colorectal neoplasia. , 2003, Molecular cancer research : MCR.

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

[16]  E. Baehrecke.  miRNAs: Micro Managers of Programmed Cell Death , 2003, Current Biology.

[17]  V. Ambros.  MicroRNA Pathways in Flies and Worms Growth, Death, Fat, Stress, and Timing , 2003, Cell.

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

[19]  C. Burge,et al.  Vertebrate MicroRNA Genes , 2003, Science.

[20]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[21]  T. Tuschl,et al.  New microRNAs from mouse and human. , 2003, RNA.

[22]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[23]  Shuguang Zhang,et al.  Design peptide scaffolds for regenerative medicine. , 2003, Advances in experimental medicine and biology.

[24]  W. Filipowicz,et al.  Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP , 2002, The EMBO journal.

[25]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[26]  J. Messing,et al.  CARPEL FACTORY, a Dicer Homolog, and HEN1, a Novel Protein, Act in microRNA Metabolism in Arabidopsis thaliana , 2002, Current Biology.

[27]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[28]  B. Reinhart,et al.  MicroRNAs in plants. , 2002, Genes & development.

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

[30]  E. Lai.  Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation , 2002, Nature Genetics.

[31]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

[32]  Thomas D. Schmittgen,et al.  Real-Time Quantitative PCR , 2002 .

[33]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[34]  T. Tuschl,et al.  Identification of Novel Genes Coding for Small Expressed RNAs , 2001, Science.

[35]  V. Ambros,et al.  An Extensive Class of Small RNAs in Caenorhabditis elegans , 2001, Science.

[36]  L. Lim,et al.  An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans , 2001, Science.

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

[38]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

[39]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.