micro RNA and qRT-PCR

220-plex microRNA expression profile of a single cell.
Tang F, Hajkova P, Barton SC, O'Carroll D, Lee C, Lao K, Surani MA.
Nat Protoc. 2006;1(3):1154-1159.
Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental
Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 days.

MicroRNA quantitation from a single cell by PCR using SYBR Green detection and LNA-based primers.
NATURE METHODS FEBRUARY 2008 sponsored by Exiqon

We describe a new, highly sensitive and specific PCR approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA microRNA PCR system. The method, which allows detection of 10 copies of miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The LNA-conferred sensitivity facilitates accurate detection of miRNA expression in a single cell.

Facile means for quantifying microRNA expression by real-time PCR.

Shi R, Chiang VL.
Biotechniques. 2005 39(4): 519-525.
North Carolina State University, Raleigh, NC 27695-7247, USA.

MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having asfew as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.

Human microRNA targets.
John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS.
PLoS Biol 2(11): e363
Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, USA.

MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3' untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.

Human microRNA targets

Drosophila microRNA targets

Zebrafish microRNA targets

miRanda software for microRNA target prediction (not including conservation analysis)

siRNAs can function as miRNAs.
Doench JG, Petersen CP, Sharp PA.
Genes Dev. 2003 17(4): 438- 442
Center for Cancer Research, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA.

With the discovery of RNA interference (RNAi) and related phenomena, new regulatory roles attributed to RNA continue to emerge. Here we show, in mammalian tissue culture, that a short interfering RNA (siRNA) can repress expression of a target mRNA with partially complementary binding sites in its 3' UTR, much like the demonstrated function of endogenously encoded microRNAs (miRNAs). The mechanism for this repression is cooperative, distinct from the catalytic mechanism of mRNA cleavage by siRNAs. The use of siRNAs to study translational repression holds promise for dissecting the sequence and structural determinants and general mechanism of gene repression by miRNAs.

Prediction and validation of microRNAs and their targets.
Bentwich I.
FEBS Lett. 2005 579(26): 5904-5910
Rosetta Genomics Ltd., 10 Plaut Street, Science Park, Rehovot 76706, Israel.

MicroRNAs are short non-coding RNAs that inhibit translation of target genes by binding to their mRNAs, and have been shown to play a central role in gene regulation in health and disease. Sophisticated computer-based prediction approaches of microRNAs and of their targets, and effective biological validation techniques for validating these predictions, now play a central role in discovery of microRNAs and elucidating their functions.

A single-molecule method for the quantitation of microRNA gene expression.
Neely LA, Patel S, Garver J, Gallo M, Hackett M, McLaughlin S, Nadel M, Harris J, Gullans S, Rooke J.
Nat Methods. 2006 (1): 41-46
US Genomics, 12 Gill Street, Suite 4700, Woburn, Massachusetts 01801, USA.

MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps. The Direct miRNA assay hybridizes two spectrally distinguishable fluorescent locked nucleic acid (LNA)-DNA oligonucleotide probes to the miRNA of interest, and then tagged molecules are directly counted on a single-molecule detection instrument. In this study, we show the assay is sensitive to femtomolar concentrations of miRNA (500 fM), has a three-log linear dynamic range and is capable of distinguishing among miRNA family members. Using this technology, we quantified expression of 45 human miRNAs within 16 different tissues, yielding a quantitative differential expression profile that correlates and expands upon published results.

A high-throughput method to monitor the expression of microRNA precursors.
Schmittgen TD, Jiang J, Liu Q, Yang L.
Nucleic Acids Res. 2004 32(4): e43
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

microRNAs (miRNAs) are small, functional, non-coding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the approximately 75 nt precursors (pre-miRNAs) by the nuclear enzyme Drosha. The approximately 22 nt mature miRNA is processed from the pre-miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors. Gene-specific primers and reverse transcriptase were used to convert the primary precursors and pre-miRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.

Real-time PCR quantification of precursor and mature microRNA.
Schmittgen TD, Lee EJ, Jiang J, Sarkar A, Yang L, Elton TS, Chen C.
Methods. 2008 Jan;44(1):31-8.
College of Pharmacy, Ohio State University, Columbus, OH 43210, USA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.

MicroRNA maturation: stepwise processing and subcellular localization.
Lee Y, Jeon K, Lee JT, Kim S, Kim VN.
EMBO J. 2002 (17): 4663-4670
Institute of Molecular Biology and Genetics and School of Biological Science,
Seoul National University, Seoul 151-742, Korea.

MicroRNAs (miRNAs) constitute a novel, phylogenetically extensive family of small RNAs ( approximately 22 nucleotides) with potential roles in gene regulation. Apart from the finding that miRNAs are produced by Dicer from the precursors of approximately 70 nucleotides (pre-miRNAs), little is known about miRNA biogenesis. Some miRNA genes have been found in close conjunction, suggesting that they are expressed as single transcriptional units. Here, we present in vivo and in vitro evidence that these clustered miRNAs are expressed polycistronically and are processed through at least two sequential steps: (i) generation of the approximately 70 nucleotide pre-miRNAs from the longer transcripts (termed pri-miRNAs); and (ii) processing of pre-miRNAs into mature miRNAs. Subcellular localization studies showed that the first and second steps are compartmentalized into the nucleus and cytoplasm, respectively, and that the pre-miRNA serves as the substrate for nuclear export. Our study suggests that the regulation of miRNA expression may occur at multiple levels, including the two processing steps and the nuclear export step. These data will provide a framework for further studies on miRNA biogenesis.

MicroRNA genes are transcribed by RNA polymerase II.
Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN.
EMBO J. 2004 (20): 4051-4060
Institute of Molecular Biology and Genetics and School of Biological Science,
Seoul National University, Seoul, Korea.

MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The primary miRNA transcripts (pri-miRNAs) contain cap structures as well as poly(A) tails, which are the unique properties of class II gene transcripts. The treatment of human cells with alpha-amanitin decreased the level of pri-miRNAs at a concentration that selectively inhibits pol II activity. Furthermore, chromatin immunoprecipitation analyses show that pol II is physically associated with a miRNA promoter. We also describe, for the first time, the detailed structure of a miRNA gene by determining the promoter and the terminator of mir-23a approximately 27a approximately 24-2. These data indicate that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. Our study offers a basis for understanding the structure and regulation of miRNA genes.

miRU: an automated plant miRNA target prediction server.
http://bioinfo3.noble.org/miRU.htm
Zhang Y.
Nucleic Acids Res. 2005 33 (Web Server issue)

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA

MicroRNAs (miRNAs) play important roles in gene expression regulation in animals and plants. Since plant miRNAs recognize their target mRNAs by near-perfect base pairing, computational sequence similarity search can be used to identify potential targets. A web-based integrated computing system, miRU, has been developed for plant miRNA target gene prediction in any plant, if a large number of sequences are available. Given a mature miRNA sequence from a plant species, the system thoroughly searches for potential complementary target sites with mismatches tolerable in miRNA-target recognition. True or false positives are estimated based on the number and type of mismatches in the target site, and on the evolutionary conservation of target complementarity in another genome which can be selected according to miRNA conservation. The output for predicted targets, ordered by mismatch scores, includes complementary sequences with mismatches highlighted in colors, original gene sequences and associated functional annotations. The miRU web server is available at http://bioinfo3.noble.org/miRU.htm

Real-time quantification of microRNAs by stem-loop RT-PCR.
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL,
Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ.
Nucleic Acids Res. 2005 33(20): e179.
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA.

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.

Profiling microRNA expression using sensitive cDNA probes and filter arrays.
Sioud M, & Rosok O.
Biotechniques. 2004 37(4): 574-580
Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway.

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.

miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.
Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, Dreyfuss G.
Genes Dev. 2002 16(6): 720-728
Howard Hughes Medical Institute, Department of Biochemistry & Biophysics,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104-6148, USA.

Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.

MicroRNA expression profiling of single whole embryonic stem cells.
Tang F, Hajkova P, Barton SC, Lao K, Surani MA.
Nucleic Acids Res. 2006 34(2): e9
Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental
Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

MicroRNAs (miRNAs) are a class of 17-25 nt non-coding RNAs that have been shown to have critical functions in a wide variety of biological processes during development. Recently developed miRNA microarray techniques have helped to accelerate research on miRNAs. However, in some instances there is only a limited amount of material available for analysis, which requires more sensitive techniques that can preferably work on single cells. Here we demonstrate that it is possible to analyse miRNA in single cells by using a real-time PCR-based 220-plex miRNA expression profiling method. Development of this technique will greatly facilitate miRNA-related research on cells, such as the founder population of primordial germ cells where rapid and dynamic changes occur in a few cells, and for analysing heterogeneous population of cells. In these and similar cases, our method of single cell analysis is critical for elucidating the diverse roles of miRNAs.

Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs.
Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM.
RNA. 2005 11(11): 1737-1744
Rosetta Inpharmatics, Seattle, WA 98109, USA.

There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.

siRNA and miRNA: an insight into RISCs.
Tang G.
Trends Biochem Sci. 2005 30(2): 106-114
Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, MA 01605, USA.

Two classes of short RNA molecule, small interfering RNA (siRNA) and microRNA (miRNA), have been identified as sequence-specific posttranscriptional regulators of gene expression. siRNA and miRNA are incorporated into related RNA-induced silencing complexes (RISCs), termed siRISC and miRISC, respectively. The current model argues that siRISC and miRISC are functionally interchangeable and target specific mRNAs for cleavage or translational repression, depending on the extent of sequence complementarity between the small RNA and its target. Emerging evidence indicates, however, that siRISC and miRISC are distinct complexes that regulate mRNA stability and translation. The assembly of RISCs can be traced from the biogenesis of the small RNA molecules and the recruitment of these RNAs by the RISC loading complex (RLC) to the transition of the RLC into the active RISC. Target recognition by the RISC can then take place through different interacting modes.

Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing.
Meister G, Landthaler M, Dorsett Y, Tuschl T.
RNA. 2004 10(3): 544-550
Laboratory of RNA Molecular Biology, The Rockefeller University, New York, USA

A large number of miRNAs have recently been discovered in plants and animals. Development of reverse genetic approaches that act to inhibit microRNA function would facilitate the study of this new class of noncoding RNA. Here we show that 2'-O-methyl oligoribonucleotides, but not 2'-deoxyoligonucleotides specifically inactivate the RNAi activity associated with miRNA-protein complexes in human cell extracts as well as in cultured human cells.

RNA silencing in plants

David Baulcombe
Nature (2004) 431, 356-363

There are at least three RNA silencing pathways for silencing specific genes in plants. In these pathways, silencing signals can be amplified and transmitted between cells, and may even be self-regulated by feedback mechanisms. Diverse biological roles of these pathways have been established, including defence against viruses, regulation of gene expression and the condensation of chromatin into heterochromatin. We are now in a good position to investigate the full extent of this functional diversity in genetic and epigenetic mechanisms of genome control.

DNA events: An RNA microcosm.

Baulcombe D.
20 SEPTEMBER 2002   VOL 297  SCIENCE
Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.

Micro-RNA (miRNA) was f irst identified as a class of regulatory RNA in animals with perhaps hundreds of different members. It appeared to represent a previously unsuspected layer of regulation in higher organisms, but from the initial reports it was not clear what was being regulated by this RNA, or how. Now a number of studies, including two by Llave et al. (page 2053) and Hutvágner and Zamore (page 2056) published in this issue are revealing that miRNAs and other similar tiny RNAs are involved at many different levels of genetic control in plants and animals.