I'd like to see if either microRNA or siRNA affects the translation of my gene of interest (protein). I understand that there are kits which detect small RNAs from total RNAs but how can I specifically look at the particular small RNAs which interferes the protein translation of my gene(s) of interest.
Thanks
Hi Arun, Thanks a lot. But my original Q was how exactly to determine which miRNA or siRNA would possibly bind and repress the the translation. Then I can clone or introduce as you had suggested. Thanks
I think you can pull down the miRISC from a sample where you know that translation of your GOI is repressed. Sequence the small RNAs from the RISC and identify the ones that have the possibility to bind with your target. Narrow your miRNA search by using software's that predict miRNAs and their targets. Then probably you can validate these miRNAs experimentally by expressing small fragments of your gene tagged to GFP in the presence and absence of miRNAs. Thanks for the nice question.
Hi Basudev,
Do you have any suggestions for a software which can guesstimate the microRNA binding probabilities in GOI?
in this you will find several suggestions
http://journal.frontiersin.org/Journal/10.3389/fgene.2014.00023/abstract
good luck
You can try Ago2 immunoprecipitation after transfection of miRNA.
Then amplify targeted genes by PCR.
Another way is PAR-CLIP.
Finally you can identify miRNA targeted genes from these experiments.
Hi,
you can do this either computationally or experimentally.
Computationally: You can visit www.microrna.gr and DIANA-microT-CDS software in particular which is an algorithm that performs miRNA target prediction. By searching the database with your gene you can find all possible miRNAs that target your gene. If you somehow (literature search, expression data etc) narrow down the candidate miRNAs, then you can perform several Reporter Gene Assays to verify the interactions.
Experimentally: Currently, the best thing to do is to perform any CLIP experiment (PAR-CLIP, HITS-CLIP, iCLIP). The advantage of this method is that you will capture all miRNA binding sites on various RNAs (coding, non-coding etc). The disadvantage is that you need to do a small RNA-Seq experiment too in order to identify the expressed miRNAs and then crossreference the two datasets to produce the final results.
In the same web portal (www.microrna.gr) there are two databases, TarBase and LncBase which have experimentally verified miRNA:gene interactions on coding and non-coding genes respectively. These interactions have been retrieved from the literature as well as from experimental data such as CLIP, Reporter Gene Assays and many more.
Hope this helps.
You should narrow choices for interaction with computational approach first. Then, you should use siRNA transfection or pull-down assay for verification of interaction. But, here selection of databases is very important. Each of them use different algorithm. You should check their working principles. For example, most of them use just 3'UTR region of genes, not exonic of 5'UTR region. Also, some of them expreimentally verified, some of them are just based on predictions.
miRWalk, STarMir, Microcosm, TargetScan, miRNAMap, mirDIP, TarMir, miR2Disease, starBase, miRo are predicted databases.
miRTarBase and DianaTools are experimentally validated miRNA databases.
Sincerely,
Sercan
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