The discovery of small noncoding RNAs capable of re-pressing, silencing, or even enhancing gene expressioncontinues to inspire researchers who study the regula-tion of gene expression. A team led by Dr. Victor Am-bros, Professor of Genetics, Dartmouth Medical School(Hanover, NH) was the first to discover microRNAs—a class of small noncoding RNAs that regulate gene ex-pression. Recently, Dr. Ambros’s laboratory has begunto apply Applied Biosystems (Foster City, CA) Taq-Man®-based (Roche Molecular Systems, Inc.,Alameda, CA) real-time PCR assays to his laboratory’sinvestigations of how microRNAs influence the fate ofcells in flies, worms, and more recently in human braincancer tumors. (TaqMan is for research use only, not foruse in diagnostic procedures.)

Through the use of real-time PCR assays, which are ca-pable of detecting and quantifying mature microRNA(miRNA) levels in different cell types, Dr. Ambroshopes to explain how miRNAs regulate the expressionof genes involved in timing pathways as cells in wormsand flies differentiate into different cell types duringdevelopment. Also, the goal of a collaboration with Dr.Mark Israel, Director of the Norris Cotton CancerCenter at Dartmouth, is to be able to assess differentpatterns of miRNA expression as a potential means fordiscerning different tumor types in cancerous humanbrain tissue samples.

Discovery of microRNAsIn 1993, Dr. Victor Ambros, Ms. Rosalind Lee, and Dr.Rhonda Feinbaum published a finding about molecularevents that transpire in the development of Caenorhabdi-tis elegans. In that paper, they also described for the firsttime the function of a small noncoding RNA genenamed lin-4 (lineage-abnormal-4). At the time, manyresearchers underestimated the enormous scientificwealth of this finding that later forged a new frontier indevelopmental biology.1

Lin-4, a gene encoding a 22-nucleotide (nt) non-coding RNA molecule that represses translation ofmRNAs into proteins involved in the timing andsequence of postembryonic development in C.elegans, was the first of a class of regulatory genesthat have come to be known as miRNAs. Since thisdiscovery, miRNAs have been found scatteredthroughout the genomes of almost all multicellularorganisms, and play a significant role in regulatinggene expression.

Small noncoding RNAsA member of a family of noncoding RNAs that includesuch relatives as small interfering RNAs (siRNAs) andshort hairpin RNAs (shRNAs), miRNAs were salvagedfrom regions of the genome that at one time were re-ferred to as junk DNA, because they consist ofstretches of bases that do not encode proteins. How-ever, despite being plentiful, Dr. Ambros notes that allnoncoding RNAs represent only a small percentage ofthe long stretches of DNA bases that lie between pro-tein coding genes.

The entire family of noncoding RNAs began to belooked at closely after 1998, the same year that CraigMello and Andy Fire published their landmark paperabout the process of RNA interference (RNAi), inwhich double-stranded RNA silences homologousgenes in an antisense fashion. A cousin of miRNAs,siRNAs are an intermediate in RNAi.2

MicroRNAs differ from siRNAs and shRNAs in boththe scope of target mRNAs they interact with and inthe mechanism of how they interfere with the transla-tion of target mRNAs into proteins. In fact, one rea-son that miRNAs intrigue researchers like Dr. Am-bros is that, in animals, they often repress translationof target mRNA molecules rather than silence themthrough directed cleavage of the transcripts.

Understanding Regulation of GeneExpression by MicroRNAs UsingReal-Time PCR Assays

by Mark Springer

Reprinted from American Biotechnology Laboratory October 2005

The characteristic hairpin structure of the ~70-nt pre-cursor miRNAs has helped researchers to discoverhundreds of different miRNAs throughout many dif-ferent cell types. To find many of these miRNAs, re-searchers have used computational methods that relyon structure-based informatics searches of noncodinggenomic sequences.

Defining cell fatesSome of the biological processes for which Dr. Am-bros’s laboratory has already characterized microRNAsinclude developmental timing processes in C. elegans.Through the study of worm and fly mutants, Dr. Am-bros’s laboratory hopes to learn how these miRNAgenes regulate biological pathways involved in the de-velopment of these animals. A detailed understandingof miRNA targets in worms and flies will also helpother researchers determine if similar targets are con-served in the analogous pathways in humans and mice.

Dr. Ambros acknowledges that it is difficult to predictthe transfer of scientific knowledge between worms,flies, and humans. He notes, however, that a completeunderstanding of the way in which stage-specific fatesin flies and worms are controlled at the molecular levelmay lead to analogous experiments that uncover timingand development pathways in human stem cells.

Refining cell typesIn addition to revealing some clues in the mystery of ani-mal development, miRNAs have also been found to havea more subtle influence on cell development, the refiningof cell types. For example, in August 2004, Dr. OliverHobert, Assistant Professor of Biochemistry and Molecu-lar Biophysics, Columbia University College of Physi-cians and Surgeons (New York, NY), described the rolesof microRNAs in establishing left–right asymmetry for asubclass of sensory neurons in C. elegans.3

In this finding by Dr. Hobert and his laboratory, theneuronal cell type is established independently of themicroRNAs, but, to make the left side of the cell dif-ferent from the right side, two different microRNAsare expressed sequentially that interfere with mRNAtranscripts for transcription factors that regulate ex-pression of genes specific for left and right chemore-ceptors. As a result, the left and right cells express dif-ferent chemoreceptor genes. Dr. Ambros notes thatmiRNAs may participate in this same kind of refiningof cell types in the vertebrate nervous system.

Detection and quantification ofmicroRNAs by real-time PCRassaysTo strive for a more comprehensive understanding ofhow microRNAs contribute to important biologicalprocesses, Dr. Ambros’s laboratory has begun to detectand quantify miRNAs using a sensitive and specific as-say from Applied Biosystems based on real-time PCRtechnology (Figure 1). One of the hallmarks of the Taq-Man-based real-time PCR assays is their ability to dis-tinguish between the hairpin structure of precursormiRNA and the short, mature miRNA molecules. Thestem-loop structure, which is specific to the 3’ end ofthe mature miRNA, presumably creates steric hin-drance to prevent priming of the precursor miRNA. Asa result, the assays detect and quantify only maturemiRNA molecules, the form capable of interactingwith target mRNA molecules. Quantification ofmiRNA levels enables researchers to compare wild-type and mutant nematodes and see more clearly howexpression of specific miRNAs regulates the expressionor function of other genes.

Until participating in a recent collaboration with Ap-plied Biosystems to investigate the use of real-time

Figure 1 TaqMan-based real-time PCR assays for quantifica-tion of microRNAs use stem-looped primers that allow a two-stepquantification of miRNAs present in a sample. In the first step,stem-looped primers anneal to target miRNAs and extend thelength of the molecule by reverse transcription-PCR (RT-PCR). Inthe second step, a real-time PCR reaction that involves a reverseprimer and a probe allows researchers to quantify the number ofmature miRNA molecules present in a sample based on fluorescentemission of a reporter dye.

PCR assays for the detection and quantitation of miR-NAs, Dr. Ambros had been primarily using Northernblotting to measure RNA levels of both mRNAs andmicroRNAs. In Northern blotting, target RNAs are de-tected by complementary DNA or RNA probes that hy-bridize to them. Northern blotting is slower, requiresmore initial RNA sample, and does not generate quan-titative data such as those resulting from real-time PCRassays that use TaqMan probes and primers. The ability

of the TaqMan-based real-time PCR assays to accuratelyquantify miRNA levels will provide data that more ac-curately describe the role of miRNAs in determiningthe fates of cells (Figure 2).

Working with small samplesAnother major advance in developmental biology re-search that Dr. Ambros anticipates will be made possi-ble by real-time PCR technology will be the labora-tory’s ability to work with small samples. He believesthat the extreme sensitivity of the TaqMan real-timePCR assays makes it possible to assay levels of specificmiRNAs in very small-sized samples of staged embryos.The results may help to uncover the roles of miRNAsin animal development.

Loose fit to targets to repressexpressionAccording to Dr. Ambros, in animals, miRNAs arethought to bind to target mRNAs primarily in the 3’untranslated region (UTR). However, in almost all in-teractions with a target mRNA, the miRNA does notbind in a tight base-to-base pairing, such as occurs be-tween the two complementary strands of DNA in adouble helix. In essentially all animal miRNA targetedinteractions, there is mismatch base pairing, and ap-parently it occurs predominantly in the 3’ UTR. Thisloose-fitting binding means that the target mRNAmolecule is not destroyed, but that the process of con-verting the mRNA message into protein sequence isinstead repressed.

According to Dr. Ambros, differences in how plantand animal miRNAs interact with their targets im-plies different mechanisms for how miRNAs interferewith translation in the plant and animal worlds. Inplants, most often microRNAs do interact with thecoding region of target mRNAs; moreover, theymatch their targets precisely. This results in target de-struction, instead of translational repression, explainsDr. Ambros.

Many questions still remain about how miRNAs inter-act with target mRNAs, or even other potential targets.However, the ability to accurately quantify bothmRNA and miRNA molecules should help to answersome of these questions.

MicroRNAs and brain cancerresearchWhile miRNAs are expressed in many different humancell types, according to Dr. Ambros, the brain cells ex-press the greatest variety of microRNAs. In a collabora-tion with Dr. Mark Israel, an oncologist who heads theNorris Cotton Cancer Center at Dartmouth, Dr. Am-bros’s laboratory has been investigating ways to use data

Figure 2 In TaqMan real-time PCR assays, the assay probecarries a fluorogenic reporter dye at its 5' end and a quencher at its 3'end. The quencher absorbs the fluorescence emission of the reporterwhile the two are in close proximity to each other as part of the probe.During each cycle of PCR, the probe hybridizes to the PCR productsto which it is targeted, and the 5' terminal reporter dye is then cleavedthrough the 5'-to-3' nuclease activity of Taq DNA polymerase as itcopies the complementary strand. The physical separation of the re-porter and quencher dyes results in an increase in fluorescent signalthat is proportional to the amount of amount of amplification productthat is generated in the reaction mixture.4

about expression of miRNA genes as a means for distin-guishing between two different kinds of brain tumors.The tumors are both astrocytomas found in one type ofglial cell. One class of tumors is not as responsive totreatment as is the other class of tumors.

The prognosis for recovery in the class of tumors re-sponsive to treatment is better than it is for the nonre-sponsive class of tumors; thus Dr. Israel and Dr. Ambrosare searching for a method or technique that can reli-ably distinguish between these two types of tumors.Traditional pathological assays and histological ap-proaches have not been useful for distinguishing be-tween these two types of tumor cells. Because miRNAsare prevalent in brain cells, the two researchers havebeen investigating the possibility that miRNAs ex-pressed in these tumor cells may serve as biomarkersthat indicate whether a particular tumor cell is the typethat is more responsive to treatment.

Results from the TaqMan real-time PCR assays of dif-ferent brain tumor types have shown, in some cases, aconsistent pattern of miRNA expression across tumorsand cell types. According to Dr. Ambros, other miR-NAs behave quite differently in different tumors.

Next, researchers in Dr. Ambros’s laboratory plan touse bioinformatics tools to relate the pattern of changesin miRNAs to those sets of predicted targets that are ei-ther present or absent in profiled tumors.

ConclusionTaqMan real-time PCR assays that quantify miRNAsgive researchers such as Dr. Victor Ambros a powerfultool for detecting and quantifying levels of miRNAs indifferent cell types. Use of these assays will enable re-searchers to connect expression of miRNAs with regu-

latory processes that ultimately determine the fates ofmany different cell types. For example, in the case ofheterochronic pathways, and left–right asymmetry inthe nervous system in C. elegans, muscle developmentin Drosophila, or even tumor formation in human braincells, whether or not specific microRNAs are expressedin a cell often influences key cellular characteristicsand functions carried out by that cell.

References1. Lee RC, Feinbaum RL, Ambros V. The C. elegans het-

erochronic gene lin-4 encodes small RNAs with antisensecomplementarity to lin-14. Cell Dec 3, 1993; 75:843–54.

2. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE,Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–11.

3. Chang S, Johnston RJ Jr, Frokjaer-Jensen C, Lockery S,Hobert O. MicroRNAs act sequentially and asymmetricallyto control chemosensory laterality in the nematode. NatureAug 12, 2004; 430(7001):785–9.

4. Livak K, Flood SJ, Marmaro J, Giusti W, Deetz K. Oligonu-cleotides with fluorescent dyes at opposite ends provide aquenched probe system useful for detecting PCR product andnucleic acid hybridization. Genome Res Jun 1995;4:357–62.

Mr. Springer is Senior Science Writer, Applied Biosystems,850 Lincoln Centre Dr., Foster City, CA 94404, U.S.A.;te l.: 650-570-6667; fax: 650-638-6239; e-mai l:SpringMN@appliedbiosystems.com.