g e n o m e t e c h n o l o g y

Manipulating MicroRNAs

a troubleshooting guide: eXPerts share their advice on

measuring micrornas

M e t h o d s

table of ContentsLetter from the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Index of Experts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Q1: How do you effectively isolate and purify miRNA

from cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Q2: How do you efficiently clone an miRNA? . . . . . . . . . 7

Q3: What method do you use to ensure sensitive and

specific gene expression profiling of miRNAs? Why? . . . 8

Q4: How do you validate your expression results so

that you get robust and reproducible data? . . . . . . . . . 10

Q5: What method do you use to up- or down-regulate

a specific miRNA? Why? . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Q6: How do you verify the results of up- or down-

regulating an miRNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

List of Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Galina GabrielyBRIgHAm ANd WomEN’s HospItAL, HARvARd UNIvERsIty

Letter from the editorThis month, GT brings

you a technical guide on manipulating microRNAs. Although the first miRNA was discovered as recently as 1993, scientists have learned much about how

these small, noncoding RNAs affect gene ex-pression. Normally, they down-regulate genes by interacting with expressed transcripts, ef-fectively turning them off or dampening their expression. Recent work has shown them to influence at least 30 percent of genes.

Not only have miRNAs been found to play an important role in regulating development as well as protecting against cancer, they are differentially expressed in tissues, making expression profiling a key area of research.

And while the toolsets for working with miRNAs — overexpressing them or knock-ing them out to see what ensues — have greatly improved during the past few years, there are still tricks to master.

To that end, we’ve consulted some of the best and brightest in the field to get feed-back on how best to perform both dis-covery and expression profiling studies. Their troubleshooting advice ranges from how to isolate and purify miRNA from cells, to how to up- or down-regulate a specific miRNA and how to verify the re-sults. We hope this steers you in the right direction. And for even more thought-ful instruction, check out our resources guide at the back.

— Jeanene Swanson

Index of expertsMany thanks to our experts for taking the time to contribute to this technical guide, which would not be possible without them.

manipulating microRNAs sEptEmBER 2009 genome technology 5

Silvia MonticelliINstItUtE foR REsEARcH IN BIomEdIcINE, sWItzERLANd

Peng JinEmoRy UNIvERsIty

Joshua MendellJoHNs HopkINs UNIvERsIty

Winston KuoBRIgHAm ANd WomEN’s HospItAL, HARvARd UNIvERsIty

manipulating microRNAs6 tech guide sEptEmBER 2009

how do you effectively isolate and purify mirna from cells?

for miRNA isolation we routinely

use tRIzol RNA reagent, which is

normally used for total RNA iso-

lation . this method is sufficient

for purification of miRNAs from

cultured cell lines . However, if

isolation is required from small

or poor-quality tissue samples,

we use the mirvana miRNA Iso-

lation kit from Ambion . With this

kit, miRNA could be isolated us-

ing either total RNA extraction

protocol or enriched for small

RNAs/miRNA .

— galina gabriely

In general we use tRIzol to iso-

late RNAs from cells . tRIzol has

been very effective to recover

small RNAs from tissues or cells .

— Peng Jin

In general, several techniques

have been tried in our lab, and

this depends on the method we

would use for profiling . tRIzol

has been very effective and the

mirvana miRNA Isolation kit

from Ambion . other methods

can also be used effectively . for

certain technologies, there is no

need for isolation or purification;

it requires the addition of a lysis

reagent . A critical issue with RNA

sample preparation is the effec-

tive precipitation of small RNA

fractions . traditional meth-

ods using 70 percent ethanol

washes do not effectively pre-

cipitate small RNAs . Increas-

ing the ethanol concentration

to >80 percent in these steps

will address this problem .

— Winston Kuo

for expression analysis in cells

and tissues, straightforward to-

tal RNA isolation methods work

well . We typically use tRIzol .

more sophisticated column-

based methods or further purifi-

cation of small RNA populations

has not been necessary in our

experience . furthermore, dNA

contamination, which can some-

times be an issue when assess-

ing mRNA abundance in tRIzol-

isolated RNA, does not seem to

be a problem when measuring

miRNAs in these samples .

If we wish to specifically

analyze small RNA populations

in cells (e .g ., for sequencing

small RNA libraries), we sim-

ply gel-purify small RNAs from

tRIzol-isolated total RNA .

— Joshua mendell

If you are not limited with the

amount of starting material (i .e .,

cells or tissues), then a regular

tRIzol extraction of total RNA is

enough, and you get good qual-

ity and reproducible yields . We

routinely use RNA purified in

this way to detect miRNAs by

northern blot and real-time pcR,

with very comparable and repro-

ducible results . It is a bit trickier

when you have smaller amounts

of starting material . you can still

just use tRIzol, but you need to

improve the RNA precipitation

step, and we found that adding

some glycogen-blue works just

fine . otherwise you can enrich

for the small-RNA fraction using

spin-column kits like the mirvana

from Ambion .

— Silvia monticelli

“For miRNA isolation we use tRIzol reagent.”

— Galina Gabriely

manipulating microRNAs sEptEmBER 2009 genome technology 7

how do you efficiently clone an mirna?

It depends on the purpose

of cloning an miRNA . If it is

for miRNA discovery or high-

throughput sequencing, we will

use 5’- and 3’-adapter ligation

to generate small RNA librar-

ies . If it is a known miRNA we

are interested in expressing,

we generally use pcR to ampli-

fy the dNA fragment contain-

ing the miRNA of interest and

then verify its expression from

an appropriate vector .

— Peng Jin

When cloning an miRNA, there

are a couple of issues . If you

are interested in cloning a large

chunk of the genomic context

surrounding the miRNA (e .g .,

upstream promoter/enhancer re-

gions), this method is often used

when endogenous transcrip-

tional regulation and processing

(e .g ., drosha/dicer action) is un-

der examination . In this instance,

standard pcR-based methods

can be used to generate miRNA

inserts with compatible restric-

tion enzyme sites for your vector

system of interest .

If you are interested in

cloning of a much smaller se-

quence encoding the miRNA

only with little or no flanking

sequence, this approach to

miRNA cloning is highly ef-

ficient . the same protocols

that are used to generate

shRNA hairpin vectors work

well for miRNA cloning . you

simply have oligos encoding

the miRNA synthesized that

contain the appropriate over-

hangs for cloning and per-

form an annealing reaction

followed by a ligation reaction

into the linearized vector . this

strategy is often used when

straight overexpression is re-

quired for the experiment and

endogenous transcription/

processing is not relevant or

even undesirable .

— Winston Kuo

to clone an miRNA-encoding

sequence from genomic dNA,

we simply pcR-amplify the pre-

miRNA hairpin and ~100 bp of

5’ and 3’ flanking sequence .

these fragments can be directly

cloned into virtually any mam-

malian expression vector (with

RNA polymerase II promoter)

and robust expression will be

achieved with rare exception .

to directly clone mature

miRNA species (e .g ., to identi-

fy novel miRNAs), small RNAs

are first isolated from total

RNA by gel purification . After

ligating on adapters, small

RNA populations are ampli-

fied by pcR and then directly

sequenced using next-gener-

ation sequencing technology

(i .e ., 454, solexa, soLid) .

— Joshua mendell

our lab doesn’t really have

much direct experience in this .

But there are many protocols

out there that are widely avail-

able and very accurate, and

from labs that have done excel-

lent work in the field . the Bartel

lab, for example, has posted on

its website very detailed proto-

cols to work on miRNAs .

— Silvia monticelli

“It depends on the purpose.”

— Peng Jin

manipulating microRNAs8 tech guide sEptEmBER 2009

What method do you use to ensure sensitive and specific gene expression profiling of mirnas? Why?

We usually perform small-scale profiling of small numbers of human miRNAs using multi-plex real-time pcR . this method provides better sensitivity and specificity than microarray profiling and therefore is pref-erable when the number of miRNAs in question is not very high . the accuracy of profiling by multiplexed Rt-pcR can be validated by more sensitive, singleplex reaction .

— galina gabriely

We generally utilize an miRNA taqman assay to determine the expression of specific miRNAs . We have tried multiple ap-proaches and found that both sensitivity and specificity of taqman are better . of course, we also use high-throughput sequencing for digital gene expression profiling of small RNAs, which has good correla-tion with taqman assays .

— Peng Jin

We have used multiple miRNA profiling technologies that in-clude those from Exiqon, febit, High throughput genomics, In-vitrogen, and Luminex . Exiqon’s

microRNA array platform that

contains probes with locked nu-

cleic acid-enhanced oligonucle-

otide capture probes . LNA are

modified nucleosides in which

the ribose ring is “locked” with

a methylene bridge connect-

ing the 2’-o atom and the 4’-c

atom . this increases the melt-

ing temperature of the duplex

2-8 degrees c per LNA mono-

mer . this vastly improves the

thermal stability and specificity

of duplexes formed with com-

plimentary miRNA sequences .

Incorporation of LNA nucleo-

sides also allows for the gener-

ation of tm-normalized capture

probes . this is very important

for the performance of the mi-

croarray, as miRNA are short

and have a broad tm range .

Luminex’s flexmiR microRNA

panels combine Exiqon’s LNA

probes to achieve high speci-

ficity . Htg, on the other hand,

uses complementary nucle-

ase protection probes that

are hybridized to the miRNA

in the sample, and then an s1

endo/exo nuclease reaction

is carried out which destroys

mismatched probes in a very

sensitive manner .

— Winston Kuo

In my laboratory, we are cur-

rently using custom-spotted

oligonucleotide microarrays

primarily because they are

cost-effective and easy to use .

for greater sensitivity, there are

good commercially-available

options such as the Exiqon and

Agilent microarrays and the

Applied Biosystems taqman

arrays (which use real-time

pcR for profiling) .

— Joshua mendell

After years of northern blots and

dot blots to profile microRNA

expression, more and more

frequently we use the miRNA

taqman assays from Applied

Biosystems, that are extremely

specific and sensitive, and the

data always correlate very well

with our northern blots .

— Silvia monticelli

“We use taqMan assays.”

— silvia Monticelli

http://www.genomeweb.com

manipulating microRNAs10 tech guide sEptEmBER 2009

how do you validate your expression results so that you get robust and reproducible data?

We use qRt-pcR with taqman microRNA assays and normal-ize the data on other miRNAs which are known to be stable . one can also use snoRNAs for normalization . In any case, we usually normalize on two to three different molecules to en-sure the robust estimation of miRNA amounts . Also, to ensure robust and reproducible data, we perform experiments with biological repeats .

— galina gabriely

By comparing different assay formats early on, we have found that miRNA taqman assay is very robust and reproducible . for the miRNAs with high abundance, we also confirm the expression data by traditional northern blot .

— Peng Jin

We validate our expression results using three compli-mentary strategies: 1 . qpcR: there are two ap-proaches we have used, the miscript syBR green system (Qiagen) and taqman miRNA assays (ABI) . We prefer Qiagen’s strategy because it involves a poly-A tailing approach that cir-

cumvents the issues presented by the observed endogenous 3’-end miRNA sequence diver-sity . this diversity is a problem for the taqman stem-loop Rt-primer strategy . from a techni-cal standpoint a single oligo dt Rt reaction (Qiagen) is superior (cost, liquid handling, variability) to the unique stem-loop Rt re-actions (taqman) that must be performed for each individual miRNA of interest . We perform qpcR assays in 384-well plates with four technical replicates/sample and three experimental replicates per treatment group (12 total wells per condition) to get robust and reproducible data . Appropriate no-template and no primer controls are included . We perform all the normal steps to optimize any qpcR assay . pilot experiments are performed to demonstrate the assays are linear (5X dilution series) and to ensure that they yield a single product (melting curve analysis) . We also perform an extensive normalization control selection experiment to ensure that 1) the normalization control does not change in our treatment groups and/or cell lines of interest and 2)

we can use the delta delta ct rela-tive quantitation method . that is the assays for our miRNA(s) of interest and the normalization control(s) need to have similar (<10 percent difference) slopes as obtained from the 5X dilu-tion series . We start with five controls (U6B snoRNA, snoR-NA24, snoRNA49, gApdH, and select the one to two most appropriate assays) . 2 . Northern blotting: We use radiolabelled LNA-based oligos as probes for miRNA northern blots . Northerns are run in triplicate (three experimental samples per treatment group) to demonstrate reproducibility . many miRNAs are found in functional-related families (share 100 percent seed sequence homology and variable 3’-end homology) . therefore, when using qpcR or northerns you must prove that under your conditions the assay only detects your miRNA of interest and not other family members . We do this by spiking synthetic mature miRNAs (Idt) into a corn (zea) RNA carrier background . for pcR each synthetic miRNA family member (in-put) is run against a panel of

continued on page 13

manipulating microRNAs sEptEmBER 2009 genome technology 11

What method do you use to up- or down-regulate a specific mirna? Why?

for down-regulation of miRNA we use antisense oligonucle-otides (Aso) modified with 2’-o-metoxyethylribose (moE) provided by our collaborators (Regulus), as they result in the most specific inhibition . In addi-tion, we use commercially avail-able locked nucleic acid (LNA)-modified oligos . due to their high-affinity binding to miRNAs, LNA-modified oligos provide strong inhibition, though they may have some off-target ef-fects . for up-regulation we use synthetic miRNA mimics avail-able through Ambion .

— galina gabriely

We use both RNA oligo- and vector-based approaches . for an RNA oligo-based approach, we apply siRNA duplex-like miRNA and antimir/antagomir to increase or block the activity of a specific miRNA . this approach is very convenient for manipulat-ing the activity of specific miRNA in cell culture . A vector-based approach has the advantages of long-term alteration and the ability to track individual cells . We utilize arti-ficial shRNA plasmids or pol II

expression vectors containing a pri-miRNA dNA fragment to overexpress a specific miRNA . to down-regulate a specific miRNA, we will either express a shRNA that could produce siRNA against the stem-loop region of an miRNA precursor or use a miRNA sponge that could express a reporter gene with multiple target sites of specific miRNA .

— Peng Jin

there are two general strategies: 1) vector/viral-based overexpres-sion of miRNAs or miRNA anti-sense sequences and 2) trans-fection of exogenous miRNA duplexes or antisense inhibitors . the method chosen depends on the design of the experiment . generally speaking, we fa-vor transfection of exogenous gain-of-function and loss-of-function reagents . vector/viral-based strategies often rely on sequential processing of the resulting transcripts by drosha/dicer and must be exported from the nucleus via Exportin 5 . miRNA processing defects at the level of drosha and dicer have been reported in established hu-

man cancer cell lines (the model systems we work in most often) . furthermore, Exportin 5 has been shown to be a bottleneck in the miRNA biogenesis pathway in cell and mouse models . Exog-enous overexpression involving dNA integration can saturate Ex-portin 5 thus competing off en-dogenous small RNA sequences . In extreme cases this can lead to cell/animal death; however, we presume that less penetrant phenotypes are also possible and could result in serious exper-imental artifacts . transfection of exogenous duplexes that enter the pathway via direct incorpo-ration into the RIsc complex cir-cumnavigate these pitfalls . of course, in cases where persistent gain-of-function or loss-of-function is required (e .g ., xenograft models of tu-mor growth) stable expres-sion using vector/viral-based solutions are required . stable lines must be established carefully, employing viral ti-tering experiments to avoid the pitfalls descibed above .

— Winston Kuo

for upregulating miRNA, we continued on page 13

manipulating microRNAs12 tech guide sEptEmBER 2009

how do you verify the results of up- or down-regulating an mirna?

to assess miRNA activity fol-lowing its manipulation, we measure expression of lu-ciferase reporter fused to the perfect binding site of the miRNA . However, the best way to evaluate the activity of miRNA is to assess the ex-pression of its natural targets (when the target of an miRNA is known) . this should be done by western blot analysis . modulation of miRNA expres-sion can be assessed by mea-suring the expression levels of miRNA using northern blot analysis . In some cases, qRt-pcR with taqman microRNA assays may also give reliable results for miRNA expression, but oligos used for miRNA modulation may interfere with primers during pcR reaction, which often affects the real expression data .

— galina gabriely

We use several strategies in-cluding miRNA qpcR, miRNA northern blot, luciferase reporter assays, mRNA target qpcR, and protein target western blot . I discussed miRNA qpcR and Northern blotting above . these

allow for direct detection of miRNA overexpression or down-regulation . Luciferase reporter assays involve cloning of 3’-UtR sequences downstream of the luciferase coding sequence . these 3’-UtRs can contain ar-tificial miRNA cognate sites or endogenous sequences from validated mRNA targets (when this information is available) . A critical control in the later strat-egy is the generation of point mutants in the seed sequence of putative cognate sites . such mutants should abrogate the ability of the miRNA to regulate luciferase expression .

— Winston Kuo

to verify miRNA up-regulation, we again use northern blotting or real-time pcR to measure miRNA abundance . measuring miRNA inhibition may not be as simple . often, antisense oligo-nucleotides do reduce the abun-dance of the miRNAs they tar-get . so one can use these same methods to monitor miRNA in-hibition . However, sequestering an miRNA in vivo may not always result in a decrease in miRNA abundance . An alternative way

to monitor miRNA inhibition is to use a reporter construct with an miRNA binding site . the miRNA will inhibit expression of the reporter unless its activity is blocked . of course, the use of such reporters is generally lim-ited to a cell culture setting .

— Joshua mendell

this is a critical step in these kinds of experiments: by trans-fecting or transducing cells you are effectively somehow changing them, so you really need a lot of controls to make sure that the effect you are seeing is really due to miRNA expression or down-regulation . It is particularly important with miRNAs because in many cases you don’t really expect a strong phenotype, but you are looking for milder effects of ‘fine-tuning’ gene expression . I think you always need to use at the very least scrambled oli-gos, miRNAs different from the one you are interested in and mutations in the seed region . I would also recommend using different approaches to try to achieve the same results .

— Silvia monticelli

manipulating microRNAs sEptEmBER 2009 genome technology 13

Q4: continued from page 10

qpcR assays for each miRNA family member (assay) . the value for the correctly paired input x assay combination is set to 100 percent relative detection and mismatched input x assay combinations are expressed relative to 100 percent . thermocycling conditions can be modified as needed to reduce assay cross-talk with other miRNA family members . In the case of northerns, synthetic miRNAs (in the car-rier RNA) for each of the miRNA family members is run on a 15 percent pAgE gel containing 8m urea . Hybridization tem-peratures are optimized so that the LNA probe only binds to the miRNA of interest and not the other family members .

once the appropriate qpcR and/or northern blotting condi-tions are established using the spike-in strategy they can be ap-plied to experimental samples . 3 . In situ hybridization: We also have used Exiqon’s dIg-labelled miRcURy LNA microRNA detection probes to confirm our miRNA expression results and tissue specificity . We developed a working in situ hybridization protocol on our frozen mouse 18 .5 d .p .c . embryonic cranial coronal sections .

— Winston Kuo

careful validation of all microar-ray results are very important, regardless of the platform used . We generally use either north-ern blotting (when we have lots of RNA to work with) or real-time pcR (when the RNA is very

precious) . We have found that both methods produce high-quality expression data when performed properly . Northern blotting is cheaper but requires a lot of RNA, whereas real-time pcR is more expensive but very sensitive .

— Joshua mendell

I am old-fashioned, I like to see bands on a gel! We make sure that expression data are always consistent using different kinds of approaches . obviously this is not always possible, as, for ex-ample, for northern blot you need 25-30 mg of total RNA per lane (versus 10 ng for the taqman-based approaches) and you just don’t always get this much RNA, especially if you are working with primary cells .

— Silvia monticelli

often construct expression vectors (in standard mamma-lian expression plasmids or viral vectors) . making these is very simple . As described above, we simply amplify the miRNA hairpin and some flanking ge-nomic sequence and clone the pcR products directly . Alterna-tively, we sometimes transiently transfect cells with synthetic miRNA mimics (RNA oligonu-cleotides identical in sequence to the mature miRNA) to over-express an miRNA . With both of these methods (plasmid vs . synthetic mimic), supraphysi-

ologic expression levels are often achieved, so care must be taken in interpreting results from these experiments . to inhibit miRNAs, we gen-erally use commercially avail-able antisense oligonucleotides . We have had success with the inhibitors made by dharmacon and Exiqon . the limitation of these reagents is that they only work transiently . to achieve stable inhibition, we have started working with so-called “miRNA sponge” constructs, as described first by phil sharp’s laboratory . these are essentially decoy transcripts with multiple

miRNA binding sites that are thought to sequester the active miRNAs in a cell .

— Joshua mendell

We use mostly lenti- or retrovirus-based transduction systems because we work mainly with primary cells and we need sus-tained expression for long periods of time . depending on the cell type, we also use transient transfec-tion with lipofectamine or Amaxa . for down-regulation we are using miRNA sponges . for short-term experiments transient transfection of antagomirs works well, too .

— Silvia monticelli

Q5: continued from page 11

manipulating microRNAs14 tech guide sEptEmBER 2009

List of resourcesOur panel of experts referred to a number of tools that may be able to help you get a handle on working with miRNAs. Whether you’re a novice or pro at manipulating microRNAs, these resources are sure to come in handy.

PubLICAtIoNschang tc, yu d, Lee ys, Wentzel EA, Arking dE, West km, dang cv, thomas-tikhonenko A, mendell Jt . Widespread microRnA repression by myc contributes to tumorigenesis . Nat genet . 2008 Jan;40(1):43-50 . Epub 2007 dec 9 .

Johnson sm, Lin sy, slack fJ . the time of ap-pearance of the c. elegans let-7 microRnA is transcriptionally controlled utilizing a temporal regulatory element in its promoter . dev Biol . 2003 Jul 15;259(2):364-79 .

krützfeldt J, Rajewsky N, Braich R, Rajeev kg, tuschl t, manoharan m, stoffel m . Silencing of microRnAs in vivo with ‘antagomirs’ . Nature . 2005 dec 1;438(7068):685-9 . Epub 2005 oct 30 .

Lagos-Quintana m, Rauhut R, yalcin A, meyer J, Lendeckel W, tuschl t . identification of tissue-specific microRnAs from mouse . curr Biol . 2002 Apr 30;12(9):735-9 .

Landgraf p, Rusu m, sheridan R, sewer A, Iovino N, Aravin A, pfeffer s, Rice A, kamphorst Ao, Landthaler m, Lin c, socci Nd, Hermida L, fulci v, chiaretti s, foà R, schliwka J, fuchs U, Novosel A, müller RU, schermer B, Bissels U, Inman J, phan Q, chien m, Weir dB, choksi R, de vita g, frezzetti d, trompeter HI, Hornung v, teng g, Hartmann g, palkovits m, di Lauro R, Wernet p, macino g, Rogler cE, Nagle JW, Ju J, papavasiliou fN, Benzing t, Lichter p, tam W, Brownstein mJ, Bosio A, Borkhardt A, Russo JJ, sander c, zavolan m, tuschl t . A mammalian microRnA expression atlas based on small RnA library sequencing . cell . 2007 Jun 29;129(7):1401-14 .

Lee Rc, feinbaum RL, Ambros v . the c. elegans heterochronic gene lin-4 encodes small RnAs with antisense complementarity to

lin-14 . cell . 1993 dec 3;75(5):843-54 .

Lim Lp, Lau Nc, garrett-Engele p, grimson A, schelter Jm, castle J, Bartel dp, Linsley ps, Johnson Jm . microarray analysis shows that some microRnAs downregulate large numbers of target mRnAs . Nature . 2005 feb 17;433(7027):769-73 . Epub 2005 Jan 30 .

marson A, Levine ss, cole mf, frampton gm, Brambrink t, Johnstone s, guenther mg, John-ston Wk, Wernig m, Newman J, calabrese Jm, dennis Lm, volkert tL, gupta s, Love J, Hannett N, sharp pA, Bartel dp, Jaenisch R, young RA . con-necting microRnA genes to the core tran-scriptional regulatory circuitry of embryonic stem cells . cell . 2008 Aug 8;134(3):521-33 .

monticelli s, Ansel km, Xiao c, socci Nd, krichevsky Am, thai tH, Rajewsky N, marks ds, sander c, Rajewsky k, Rao A, kosik ks . microRnA profiling of the murine hematopoietic system . genome Biol . 2005;6(8):R71 . Epub 2005 Aug 1 .

vella mc, choi Ey, Lin sy, Reinert k, slack fJ . the c. elegans microRnA let-7 binds to imperfect let-7 complementary sites from the lin-41 3’utR . genes dev . 2004 Jan 15;18(2):132-7 . Epub 2004 Jan 16 .

WebsItesmiRBase http://microrna .sanger .ac .uk/sequences

microRnA.org http://www .microrna .org/microrna/home .do

target gene Prediction at emBl http://www .russell .embl .de/miRNAs

CoNFeReNCesmicroRnA and cancer keystone symposia on molecular and cellular Biology

microRnA in human disease & development cambridge Healthtech Institute

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