Huh7.5.1 cells grown in 96-well white microplates (in 5 replicates) were transfected with various miRNA mimics (at 25 nmol/L) or SMARTpool siRNAs (at 50 nmol/L). After 72 h, cells were infected with HCV P7-Luc (an HCVcc system encoding a Renilla luciferase reporter, provided by C. Rice of The Rockefeller University, New York, NY). At 48 h post-infection, cell lysates were prepared and analyzed for Renilla luciferase activities per manufacturer’s instructions (Promega) using a POLARstar multidetection microplate reader (BMG Labtech).
Polarstar multidetection microplate reader
Manufactured by BMG Labtech
The POLARstar multidetection microplate reader is a versatile instrument designed for the analysis of various biological samples in microplate format. It offers detection of multiple detection modes, including absorbance, fluorescence, luminescence, and Alpha Technology. The POLARstar is capable of performing a wide range of assays and is suitable for a variety of applications in life science research and drug discovery.
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4 protocols using polarstar multidetection microplate reader
1
HCV Infection Assay in Huh7.5.1 Cells
2
Profiling miRNA's Effect on Viral Replication
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Huh7.5.1 cells were treated with the mimic control, miR-135a or miR-122 mimic (at 25 nmol/L) for 72 h in 96-well white plates (in 5 replicates), and then transfected with JFH1-RLuc subgenomic replicon RNA (provided by C. Rice of The Rockefeller University, New York, NY) using DMRIE-C (Thermo Fisher Scientific). Cell lysates were collected after 48 h and measured for Renilla luciferase activities per manufacturer’s instructions (Promega) using a POLARstar multidetection microplate reader (BMG Labtech).
3
miRNA-mediated Modulation of HCV IRES
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Huh7.5.1 cells were transfected with various miRNA mimics as described above and incubated for 72 h. Cell were then transfected with pHCV-CLX-CMV RNA (encoding HCV IRES that directs the translation of a firefly luciferase reporter gene, provided by M. Niepmann of Giessen University, Giessen, Germany). After 24 h, cell lysates were obtained and firefly luciferase activity was subsequently measured per manufacturer’s instructions (Promega) using a POLARstar multidetection microplate reader (BMG Labtech).
4
Validating miR-135a Target Genes
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The LightSwitch 3′ UTR reporter GoClone constructs of various putative miR-135a targets were purchased from SwitchGear Genomics (Active Motif). Huh7.5.1 cells were transfected with miR-135a WT or MUT or with mimic control in 96-well white plates (in 5 replicates). After 24 h, cells were further transfected with 50 ng of GoClone 3′ UTR reporter plasmid using FuGENE 6 transfection reagent (Roche). Two days later, cells were lysed and total luciferase outputs were quantified using LightSwitch Luciferase Assay Reagent, according to the manufacturer’s instructions (SwitchGear Genomics) using a POLARstar multidetection microplate reader (BMG Labtech).
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