Microrna Target Validation
Microrna Target Validation - Related Videos
10:48
Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes
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Cited by 10 •
2015
With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.
07:19
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
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Cited by 22 •
2011
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
01:22
MicroRNAs
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2019
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
09:05
MicroRNA-based Regulation of Picornavirus Tropism
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Cited by 4 •
2017
We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.
07:23
Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome
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Cited by 3 •
2016
Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.
FAQs
A reporter provides a measurable output that can change when the introduced microRNA interacts with the linked target sequence. Researchers can compare reporter activity after microRNA introduction or inhibition, connecting a predicted interaction to a functional effect. Using a sequence from an mRNA 3′ untranslated region keeps the test focused on a candidate regulatory site.
Mutating the predicted binding site tests whether the observed reporter change depends on that particular sequence rather than on unrelated effects of microRNA treatment. If regulation changes for the original target sequence but is lost or altered after mutation, the comparison supports sequence-specific control. This makes the mutation a critical specificity test within the validation strategy.
Reporter activity tests a candidate site in a construct, whereas quantitative PCR examines effects on endogenous transcripts and protein analysis assesses translation-related consequences. Together, these readouts connect site-level regulation with changes in the natural gene context and with downstream protein production. That combination helps clarify how a microRNA may influence a cellular regulatory network rather than relying on one measurement alone.
Researchers begin with a predicted microRNA binding site, commonly from an mRNA 3′ untranslated region, and place the corresponding target sequence downstream of a reporter gene. They then introduce the microRNA or inhibit it and measure reporter activity. A construct carrying a mutated binding site provides the sequence-specific comparison needed to interpret the result.
After reporter testing, quantitative PCR can assess whether microRNA treatment is associated with changes in the endogenous target transcript, while protein analyses examine effects on translation. These measurements move the investigation from an engineered target sequence toward the gene’s natural cellular context. Concordant results across reporter, transcript, and protein readouts can strengthen interpretation of regulatory effects.
It helps connect post-transcriptional regulation with cellular function by testing candidate links within gene regulatory networks. The approach is relevant to biological studies of development, disease, and therapeutic research, where researchers need to determine whether predicted microRNA interactions have measurable effects on transcripts or translation. Its value lies in linking a specific binding site to broader cellular questions.