Gene regulation with miRNA: mechanisms, implications, techniques

• Paula Mello

  Courses...

  I'm looking for a microRNA or ncRNA short course. Does anyone knows about it?
• Rick Anthony Ricketson

  I have a potential novel miRNA from ebolavirus. How may this molecule be processed in the cytoplasm?

  5' UTR of the nucleoprotein gene (genomic) from ebolavirus (Pandora Element) showing miRNA precursor (ab initio). No pseudoknot as seen in Reston virus (RESTV).

  

  Recent replies ⋅ Show All (15)

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    Rick Anthony Ricketson replied

    There is a 10-mer match to hsa-miR-2054 in the sequence of the terminal hairpin in case you need this information which targets TNF and IL-1 for example.

    2 days ago
• Nandula Vardhini

  Apart from qRT-PCR what are the other assays which can be performed to support gene expression?

  Apart from qRT-PCR what are the other assays which can be performed to support gene expression?

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    Jon Moulton replied

    For some proteins in may be possible to assess an enzymatic activity.

    5 days ago
• Veena Rawat

  Can any one help me?

  I m doing data mining of miR-181a in my project. I have found the validated targets, snps, fragile site and possible pathways of miR-181a, but i dont understand how to elaborate my project by usingI m doing data mining of miR-181a in my project. I have found the validated targets, snps, fragile site and possible pathways of miR-181a, but i dont understand how to elaborate my project by using this information.

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    Manvendra K Singh replied

    Hi Veena, What system you are using for this?? Its good that you have validates your targets so you can come up with a hypothesis and then test it in your desired model system. You have to show howHi Veena, What system you are using for this?? Its good that you have validates your targets so you can come up with a hypothesis and then test it in your desired model system. You have to show how miR-181a regulates X,Y, Z, functions to make it a significant story. good luck Manav

    6 days ago
• Muhasin Asaf

  Discrepancies between deep sequencing and qRT-PCR results

  Can anybody please troubleshoot why my results based on differential expression of deep sequencing and qRT-PCR are not tallying. A list of miRNAs were found to be upregulated by deep sequencingCan anybody please troubleshoot why my results based on differential expression of deep sequencing and qRT-PCR are not tallying. A list of miRNAs were found to be upregulated by deep sequencing approach (SOLiD platform) but when done with qRT-PCR, they were found to be downregulated.

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    Muhasin Asaf replied

    Thank you all for your replies. I could get the results. Though the fold change in NGS and qRT-PCR are not the same but the trend was found to be same.

    9 days ago
• Miguel Torres-Martin

  Should we see mRNA downregulation caused by microRNA upregulation?

  I understand that microRNAs (usually) blocks specific mRNAs and that protein level would be decreased, but I would like to know, if we should detect mRNA downregulation by qRT-PCR in a specificI understand that microRNAs (usually) blocks specific mRNAs and that protein level would be decreased, but I would like to know, if we should detect mRNA downregulation by qRT-PCR in a specific microRNA-upregulation context, or by the contrary, the mRNA detected should not be affected by this. Any paper supporting? Thanks.

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    Matteo Marzi replied

    Yes, but the fold change can range from as little as 20% decrease to a maximum of 2/3 fold change. Be aware that a time course of the modulation of the target mRNA by your miRNA might be important toYes, but the fold change can range from as little as 20% decrease to a maximum of 2/3 fold change. Be aware that a time course of the modulation of the target mRNA by your miRNA might be important to dissect direct and indirect effect on gene expression. A direct target should be repressed at early time points. Later, you are going to face also the phenotype caused by miRNA over-expression, if anyl

    10 days ago
• Kyle Alpha

  Forgot the name of a miRNA target search, does anybody know what the site is I'm trying to remember?

  A few weeks ago, I found a great site that combined several (at least 3 or 4) miRNA target prediction algorithms, and let you search either by miRNA or by the target gene. I really want to continueA few weeks ago, I found a great site that combined several (at least 3 or 4) miRNA target prediction algorithms, and let you search either by miRNA or by the target gene. I really want to continue using it, but now I can't find it. Does anyone know the name of the site? I vaguely remember a red background.

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    Harsh Dweep replied

    miRWalk integrates 9 miRNA-target prediction programs for the comparison of miRNA binding sites within mRNA 3UTR region. miRWalk has been referenced in more than 23 articles within in one year. YoumiRWalk integrates 9 miRNA-target prediction programs for the comparison of miRNA binding sites within mRNA 3UTR region. miRWalk has been referenced in more than 23 articles within in one year. You can visit it here http://mirwalk.uni-hd.de/

    11 days ago
• Raúl Montañez Martínez

  Mechanistic description of LNA inhibition

  I'm working in a dynamical model of microRNA inhibition and their behavior under different experimental conditions. Does any one know how the LNA works? Is a thermodynamic competition with theI'm working in a dynamical model of microRNA inhibition and their behavior under different experimental conditions. Does any one know how the LNA works? Is a thermodynamic competition with the target afther the AGO loading, Is a interference in the AGO loading process. Is a interference in the hairpin structure. Each mechanism have a different consequence in the global equilibrium and is modeled in a different way. Can anyone help me?

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    Raúl Montañez Martínez replied

    Hi, What do you think about this idea? The main interference in the microRNA pathway by LNAs is blocking the AGO-microRNA complex. Could be the AGO-miRNA complex sequestrated by the LNA highly stableHi, What do you think about this idea? The main interference in the microRNA pathway by LNAs is blocking the AGO-microRNA complex. Could be the AGO-miRNA complex sequestrated by the LNA highly stable duplex? I was thinking a lot about this, because the levels of the microRNA are low when you was treated the organism with the LNAs, but really, you can not measure the levels of the miRNAs in your samples because the melting temperature of the LNAs hetero-duplex are higher that the temperature that you use in the adaptor binding and the retro-transcription processes, 75ºC.

    19 days ago
• Marilena D. Papaioannou

  Cloning the entire length of a predicted 3'UTR

  I am having some problems cloning a big 3'UTR for luciferase assays. This particular 3'UTR is 4.6kbs in length, and I cannot clone it after several attempts. Do you think that if I clone a smallI am having some problems cloning a big 3'UTR for luciferase assays. This particular 3'UTR is 4.6kbs in length, and I cannot clone it after several attempts. Do you think that if I clone a small fragment of it (obviously containing the predicted binding site for my miR of interest) would be ok? I have doubts about this, because I am thinking that perhaps this particular miR binding site could be, for example, functional only when some secondary structure of the 3'UTR forms (in the presence of other miR binding sites of the 3UTR. Which would mean that if i leave them out, I might not be recapitulating the true miR-mRNA effect. Any thoughts/suggestions?

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    Vlady Vladimirov replied

    Hi, I just wanted to add one more comment. The luc assays are really to confirm that given miRNA does bind tSpecifically to it's respective target sequence, nothing more. You're right that theHi, I just wanted to add one more comment. The luc assays are really to confirm that given miRNA does bind tSpecifically to it's respective target sequence, nothing more. You're right that the presence of the entire 3'UTR might affect the binding, but unless you have prior evidence (e.g. RNA folding, however inaccurate they might be) that access to the target sequence is hampered by secondary structures, if the miRNA binds to the target you should see some effect. It might be that the effect in the whole 3'UTR is different (either stronger or weaker) compared to the short sequence only.

    21 days ago
• Kyle Alpha

  How can I check a miRNA promoter for transcription factor binding sites?

  I'm trying to find out if the transcription factor LEF-1 binds to certain miRNA promoters, but I'm having trouble. I can't really seem to find any information on miRNA promoters OR on LEF-1'sI'm trying to find out if the transcription factor LEF-1 binds to certain miRNA promoters, but I'm having trouble. I can't really seem to find any information on miRNA promoters OR on LEF-1's recognition sequence. I've tried using Eponine to find the miR transcription start sites and promoters, but I'm new with ensemble and eponine, and can't really figure it out. Any advice on finding the miR promoters, the LEF-1 recognition site, and then putting it together? Thanks!

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    Jun He replied

    Hi, The first thing you need to know is the DNA binding motif of LEF-1. Then, you can search this consensus sequence in the promoter region of the miRNA. As you don't know where the promoter regionHi, The first thing you need to know is the DNA binding motif of LEF-1. Then, you can search this consensus sequence in the promoter region of the miRNA. As you don't know where the promoter region is, you may search up to 5kb upstream of TSS (UCSC). If you can find several binding sequence, go ahead to do ChIP assay: Design the primers covering binding sites; Use LEF-1 antibody to pull down the chromatin-DNA complex which will be template for PCR; Do PCR and run gel. If you can amplify the DNA fragment with binding sites, you may also construct miRNA promoter luciferase reporter to further validate it.

    22 days ago
• Sebastian Vencken

  In a luciferase reporter/pre-miR co-transfection experiment, how can I compensate for competitive effects from the endogenous mRNA

  I'm trying to validate a specific target of a specific miRNA by transfecting my cells by upregulating this miRNA with synthetic pre-miRs and using the 3'UTR sequence cloned with luciferase reportersI'm trying to validate a specific target of a specific miRNA by transfecting my cells by upregulating this miRNA with synthetic pre-miRs and using the 3'UTR sequence cloned with luciferase reporters to assess whether the said miRNA targets this sequence. My worry with this experiment is that the endogenous target mRNA, which is highly expressed, is competing with the reporter. Is this a common occurrence and would anybody have any experience on how to resolve this? It has to be said that the miRNA is quite strongly upregulated.

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    Sebastian Vencken replied

    @ Paul and Sanka. I've found similar results as you, the upregulation of the miRNA is indeed very high. Although the likelihood that competitive inhibition from endogenous mRNA is reduced by this, I@ Paul and Sanka. I've found similar results as you, the upregulation of the miRNA is indeed very high. Although the likelihood that competitive inhibition from endogenous mRNA is reduced by this, I was seeking to rule out this possibility.

    27 days ago
• Jon Moulton

  New paper -- Anne did some fine work here! Lagendijk AK, Moulton JD, Bakkers J. Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues.New paper -- Anne did some fine work here! Lagendijk AK, Moulton JD, Bakkers J. Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biology Open. 2012;[Advance Online Publication] doi:10.1242/bio.2012810

  http://bio.biologists.org/content/early/2012/04/11/bio.2012810.full
• Jesus Lozano

  Questions about miRNA mimics via microinjection in arthropods (in vivo)

  miRNA antagonists (i.e. LNA) have been shown as a very efficient technique to downregulate the expression of microRNA in insects, via hemocele injection. Does anyone knows if the same technic wasmiRNA antagonists (i.e. LNA) have been shown as a very efficient technique to downregulate the expression of microRNA in insects, via hemocele injection. Does anyone knows if the same technic was used with miRNA mimics? If yes, was it done with precursors to miRNA, double strand mimics or single strand mimics?

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    Harald Osmundsen replied

    Unlikely in my view. You are here looking for a sequence-specific effect. Your controls will be injections of a corresponding RNA molecule, but with a scrambled sequence.

    Apr 25, 2012
• Marilena D. Papaioannou

  What if the 3'UTR of a predicted target I want to test with Luciferase assays has internal polyA signals?

  Hi all, I am trying to clone a big 3'UTR(~3kb) of a gene that is predicted to be a target for my miRNA of interest. When I scan through the 3'UTR sequence I retrieve from Ensemble, I can locateHi all, I am trying to clone a big 3'UTR(~3kb) of a gene that is predicted to be a target for my miRNA of interest. When I scan through the 3'UTR sequence I retrieve from Ensemble, I can locate within it (and most importantly, before the predicted miRNA binding site) several polyA signals. Do you think this is a problem?

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    Marilena D. Papaioannou replied

    hi robert, thanks for your answer. these are to be used for in vitro luciferase assays. i am indeed making a mutated (in the appropriate seed seq) construct for every 3UTR i will use. i will checkhi robert, thanks for your answer. these are to be used for in vitro luciferase assays. i am indeed making a mutated (in the appropriate seed seq) construct for every 3UTR i will use. i will check out the papers you mention, thank you again!

    Apr 24, 2012
• Aruna Govindaraju

  Dnase treatment

  I am isolating total RNA by Trizol method to be used for miRNA qPCR analysis. Can you please suggest some DNase treatment methods and inactivation methods? p.s. I know EDTA and heat inactivation willI am isolating total RNA by Trizol method to be used for miRNA qPCR analysis. Can you please suggest some DNase treatment methods and inactivation methods? p.s. I know EDTA and heat inactivation will inactivate DNase. But my concern is EDTA may affect PCR and heat inactivation may degrade small RNAs. Any suggestions?

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    Tamas Kaucsar replied

    @ Sihan Wu: "Don't worry about the DNA if you use stem-loop method for qPCR. It is quite specific." I thought the same way until I checked. I've got different results with and without DNase treatment@ Sihan Wu: "Don't worry about the DNA if you use stem-loop method for qPCR. It is quite specific." I thought the same way until I checked. I've got different results with and without DNase treatment using the miRNA assay from AB, LifeTech based on stem-loop primers. "Also, you won't get any DNA if you don't touch the red organic layer below after chloroform added and centrifuged." Theoretically yes. Practically no. I transfer cca. 80% of the aqueous phase, carefully not to touch the interphase (I leave a layer of clear aqueous phase on the organic one). Even so on gel electrophoresis I see the genomic DNA contamination band. After TURBO™ DNase treatment it disappears. I usually re-extract RNA (efficiency around 70%), because qPCR results are more reproducible this way (compared to EDTA stop).

    Apr 20, 2012