Modulating miR-330 Expression In Vitro

A miRNA-suppressing miR-ZIP plasmid was used for in vitro miR-330-5p suppression (catalogue number MZIP-330-5p-PA-1, System Biosciences, California, USA) as previously described [20 (link)]. Cells were transfected with the miR-ZIP plasmid or a scrambled non-targeting vector control MIRZIP-VC plasmid (catalogue number MZIP000-PA-1, System Biosciences) using Lipofectamine 2000 (Invitrogen, ThermoFisher Scientific, UK). The single clone (SC) cell line was derived from an individual clone that was selected after assessing GFP expression using fluorescent microscopy. The SC cell line had high levels of GFP expression indicating high expression of the miRZIP-330-5p plasmid. The miRZIP-VC SC was derived from an individual clone that was selected after assessing GFP expression using fluorescent microscopy. The heterogeneous clonal (HC) cell line was derived from a mixed population of stable clones. The miRZIP-VC HC was derived from a mixed population of stable clones. The miR-330 precursor plasmid was used for in vitro miR-330-3p/5p overexpression (catalogue number PMIRH330PA-1, System Biosciences). The miR-VC (catalogue number CD511B-1, System Biosciences) vector control plasmid was used as a control. Transient overexpression of miR-330-3p and miR-330-5p was confirmed via qPCR analysis, as previously described [20 (link)].

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Bibby B.A., Miranda C.S., Reynolds J.V., Cawthorne C.J, & Maher S.G. (2019). Silencing microRNA-330-5p increases MMP1 expression and promotes an invasive phenotype in oesophageal adenocarcinoma. BMC Cancer, 19, 784.

Publication 2019

MZIP-330-5p-PA-1 MZIP000-PA-1 Lipofectamine 2000

Cell line Cells Clones Heterogeneous Lipofectamine 2000 Microscopy Mirna Plasmid Transient

Corresponding Organization :

Other organizations : University of Hull, Trinity College Dublin, St. James's Hospital, KU Leuven

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Variable analysis

independent variables

MiR-ZIP plasmid for miR-330-5p suppression
Scrambled non-targeting vector control MIRZIP-VC plasmid
MiR-330 precursor plasmid for miR-330-3p/5p overexpression
MiR-VC vector control plasmid

dependent variables

GFP expression levels (indicating miRZIP-330-5p plasmid expression)
MiR-330-3p and miR-330-5p expression levels (confirmed via qPCR analysis)

control variables

Lipofectamine 2000 transfection reagent
Cell lines: single clone (SC) and heterogeneous clonal (HC)

positive controls

MiRZIP-VC SC cell line
MiRZIP-VC HC cell line

negative controls

MiR-VC vector control plasmid

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