AVAILABLE PRODUCTS

miRNA & 3’ UTR Luciferase Construct Collection

genome-wide collection of pre-cloned luciferase 3´UTR reporters

The LightSwitch™ collection includes over 12,000 human endogenous 3`UTRs and hundreds of miRNA target bionsensors cloned into LightSwitch reporter vectors. These transfection-ready LightSwitch GoClones® together with the highly optimized assay reagents, transfection reagents, controls, miRNA Mimics and Inhibitors and detailed protocols allows you to perform your gene regulation reporter assay experiments immediately.

To ensure high-quality results, LightSwitch reporter vectors utilize a novel, engineered luciferase gene (RenSP) and optimized reagents that provide industry-leading sensitivity. For more information, please see the tabs below as well as the Overview and RenSP Luciferase sections on the Functional Genomics - LightSwitch™ page.

Click the button below to search the LightSwitch Reporter Collections and place your order on our affiliate website, SwitchGear Genomics.

  Search The Promoter &
3' UTR GoClone™ Collections

Note: All LightSwitch constructs MUST be used with the LightSwitch Luciferase Assay Reagents to obtain optimal results with our novel RenSP luciferase. We also recommend that you include appropriate positive & negative control vectors when you perform your assays.

 

Getting Started with your LightSwitch 3`UTR Assay

Post transcriptional regulation through miRNA-3´UTR interactions

Gene regulation studies have historically focused on events that influence transcription, such as the binding of transcription factors to promoters. More recently, much research has centered on the presence or absence of histone modifications that alter the chromatin structure, which influences DNA accessibility and its ability to be transcribed. However, post-transcriptional events play an equally important role in gene regulation.

MicroRNAs (miRNAs) are small, non-coding RNA molecules of approximately 22 nucleotides that play a large role as post-transcriptional regulators. miRNAs are thought to repress expression by targeting the 3´UTR (untranslated) region of mRNA transcripts. The interaction of a miRNA with a 3´UTR results in either increased degradation of the target transcript or inhibition of translation.

While the human genome is thought to encode only about 1000 miRNAs, they are believed to target ~60% of mRNA transcripts. A given miRNA may have multiple 3´UTR targets, and a given 3´UTR may be targeted by multiple miRNAs. Therefore, there is considerable interest in studying miRNA-3´UTR interactions.

LightSwitch products for the study of 3´UTR-miRNA interactions

LightSwitch 3´UTR Reporter constructs are ideal for performing miRNA target validation, as well as assessing the functional impact of miRNA-3´UTR interactions. In combination with our miRNA Mimics and Inhibitors, which are co-transfected with the 3´UTR Reporter constructs (Figure 1), you have everything needed to validate miRNA targets, measure RNA stability, translation efficiency and the functional impact of miRNAs on a gene-by-gene basis. We also offer over 900 optimized LightSwitch Synthetic miRNA Target Reporter constructs, which can provide increased response relative to the endogenous sequences cloned into the LightSwitch 3´UTR Reporter constructs.

Diagram depicting an experiment using a LightSwitch 3´UTR Reporter vector

Figure 1: Luciferase activity used for target validation of miRNAs on a specific human 3´UTR.

  1. Transfect a 3´UTR Reporter construct into your cell line.
  2. A constitutive promoter drives the production of a hybrid luciferase-human 3´UTR transcript.
  3. Total luciferase output depends on the effect of the 3´UTR on transcript stability and/or translational efficiency.
  4. A synthetic microRNA or microRNA inhibitor can be co-transfected with the 3´UTR reporter vector to study its impact.

The 3´UTR activity can be measured to quantitively characterize:

  • The impact of the UTR on post-transcriptional gene regulation
  • The impact of a miRNA or siRNA on the regulation of transcript stability or translation efficiency
  • The targets of a miRNA or siRNA (Figure 2)
  • The effect of sequence variants on UTR function
  • The impact of miRNA inhibitors or stimuli
Graph showing dose response of a LightSwitch SYT8 Promoter Reporter vector stimulated by 5 different estrogen compounds
Figure 2: Predicted mir-122 targets were validated using constructs from the LightSwitch 3´UTR Reporter Collection.

Putative 3´UTR targets of miR-122 that had been predicted using the miRanda algorithm were co-transfected with either 20 nM of miR-122 mimic or a non-targeting control miRNA in K-562 cells. The graph above shows the four strongest responders from this experiment along with two non-responding controls.

Applications

  • Understand the mechanism by which a gene is induced or repressed.
  • Measure the impact of the 3´UTR on post-transcriptional gene regulation.
  • Validate the targets of a miRNA or siRNA.
  • Measure the effects of sequence variants on 3´UTR or miRNA function.

The LightSwitch 3´UTR Reporter Collection includes over 12,000 human 3´UTRs that have been cloned into the pLightSwitch_3UTR reporter vector, which utilizes the novel, engineered RenSP luciferase gene. LightSwitch 3´UTR Reporter constructs can be used with the optimized reagents found in the LightSwitch Luciferase Assay Kit to measure the impact of a 3´UTR on post-transcriptional regulation.

In addition to the over 12,000 endogenous human 3´UTR clones that make up the LightSwitch 3´UTR Reporter Collection, we offer over 900 LightSwitch Synthetic miRNA Target Reporter constructs. These constructs, which have also been cloned into the pLightSwitch_3UTR vector, contain optimized sequence repeats, so can provide better sensitivity and a stronger response than their native counterparts.

pLightSwitch_3UTR vector diagram

Both the LightSwitch 3´UTR Reporter constructs and the LightSwitch Synthetic miRNA Target Reporter constructs were designed to be co-transfected with our collections of LightSwitch miRNA Mimics and Inhibitors to study the effects of over-expression of a miRNA of interest, or to knock down the expression of endogenous human miRNAs in living cells.

The following publications cite the use of and/or provide additional information about the LightSwitch Luciferase Assay System:

3´UTRs & miRNAs