Genome-wide Collection of Lenti 3’UTR Reporters for Functional Validation of microRNA Targets
Sigma® Life Science and SwitchGear Genomics™
Introduction
microRNAs (miRNAs) are a class of naturally occurring small non-coding RNA molecules that regulate a variety of developmental and physiological processes. To better understand miRNA-UTR interactions, Sigma Life Science has partnered with SwitchGear Genomics to create a genome-wide collection of 10,000 3’ UTR lenti-based luciferase reporters. This collection can be used to validate miRNA targets identified by prediction algorithms or other experimental methodologies.
Here we present data on a screen of a large set of predicted miR-122 targets using 3’ UTR luciferase GoClone™ reporters and validation of several known mRNA targets of human miRNAs using the MISSION® 3’UTR Lenti Goclones and microRNA mimics.
Figure 1.SwitchGear Genomics has made an extensively validated set of 3’UTR plasmid based constructs. This genome-wide set has been a proven tool for screening and validation of many different gene targets. Above is data from a screen of a large set of predicted miR-122 targets using 3’ UTR luciferase GoClone reporters and identified several novel targets. Recently, this same 3’UTR content has been used to create a genome-wide library in lentivirus format
Repression of Lenti GoClone™ Activity Using miRNA Mimics with Published Human miRNA Targets
Figure 2.microRNA Mimics Knockdown Reporter Activity of Stable MCF7 Cells Transduced with Lenti GoClones. Stable MCF7 cells transduced with Lenti GoClones were transfected with a final concentration of 50nM MISSION® Human microRNA Mimics, in parentheses (red bars) and with MISSION miRNA Negative Control 1 (white bars). Twenty-four hours post-transfection cells were assayed for Renilla luciferase activity using the LightSwitch™ Assay System on a SpectraMax® L Microplate Reader. This was done with four replicates, and the values were plotted as a percent expression compared to GoClone plus negative control as 100 percent expression
Stable Cell Line Creation with MISSION 3’UTR Lenti GoClone Workflow —a Renewable Resource for miRNA Validation
Figure 3.Renilla Luciferase Reporter Activity Detected in Stable MCF7 Cells Transduced with Lenti GoClones. Puromycin-resistant MCF7 cells were assayed for Renilla luciferase activity using the LightSwitch™ Assay System on a SpectraMax® L Microplate Reader. This was done with four replicates and luciferase output plotted as a value of relative luciferase units (RLU)
MISSION 3’UTR Lenti GoClones Respond Specifically to hsa-miR-122 Validated Gene Targets
Figure 4.MISSION 3’UTR Lenti GoClones were assessed for specificity and performance by analyzing previously identified and validated targets for hsa-miR-122. HT1080 cells were transfected with Lenti GoClones for RIMS1, GNDPA2, LUZP1, or non-targets for hsa-miR-122, which included two random 3’UTR sequences, GAPDH, and vector containing no insert. 24 hours after transfection, cells were treated with 50nM MISSION miRNA mimic for hsa-miR-122 or MISSION miRNA Negative Control 1. Knockdown of the target was measured by taking the log2 of miR-122 mimic/non-targeting control
Conclusions
- Validated miRNA target knockdown in both plasmid and lentiviral-based UTR reporter constructs
- MISSION 3’ Lenti GoClones allow integration into cell lines recalcitrant to transfection and the creation of stable 3’UTR reporter cell lines
- Genome-wide pre-cloned 3’UTR sequences provide researchers measurable savings in both time and money
Materials
©2012 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. MISSION is a registered trademark of Sigma-Aldrich Co. LLC. SwitchGear Genomics, LightSwitch and GoClone are trademarks of SwitchGear Genomics. SpectraMax is a registered trademark of Molecular Devices Corp.
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