miRNA Reporter Vector Construction

To generate reporter vectors bearing binding sites for the three miRNA examined in detail, miR-98, miR-212 and miR-137, oligonucleotides encoding target gene miRNA recognition elements (MREs) were annealed to form SpeI and HindIII restricted overhangs of a ligatable cassette compatible with SpeI and HindIII digested pMIR-REPORT vector (Ambion, Austin, TX) as described previously
[69 (link), 70 (link)]. Recombinant reporter constructs were amplified in chemicompetent DH5alpha Escherichia coli bacteria cells (Bioline, Sydney) and purified using endotoxin free minipreps (Promega, Madison, WI) before being verified by DNA sequencing (AGRF, St. Lucia, QLD, Australia) and quantitated using a NanoDrop 2000 (Thermo Scientific, Delaware, ME, USA).

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Hollins S.L., Goldie B.J., Carroll A.P., Mason E.A., Walker F.R., Eyles D.W, & Cairns M.J. (2014). Ontogeny of small RNA in the regulation of mammalian brain development. BMC Genomics, 15(1), 777.

Publication 2014

pMIR-REPORT vector DNA sequencing NanoDrop 2000

Austin Bacteria Bacteria coli Binding sites Cells Endotoxin Escherichia Gene elements Mir 137 Mirna Oligonucleotides Promega Vector

Corresponding Organization :

Other organizations : University of Newcastle Australia, University of Queensland, Queensland Centre for Mental Health Research

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Variable analysis

independent variables

Oligonucleotides encoding target gene miRNA recognition elements (MREs)

dependent variables

Reporter gene expression

control variables

PMIR-REPORT vector (Ambion, Austin, TX)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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