Wallac 1420 luminometer

A luciferase reporter assay was carried out in this study to study the regulatory relationship between PTEN promoter and LINC00470. In brief, the wild‐type PTEN promoter containing the LINC00470 binding site was cloned into a pMIR‐REPORT luciferase vector (Ambion). At the same time, site directed mutagenesis was carried out using a Quick Change II mutagenesis kit (Stratagene) based on the directions provided by the assay kit manufacturer on the product manual to induce a single site mutation in the LINC00470 binding site of PTEN promoter, and the mutant PTEN promoter sequence was also cloned into the pMIR‐REPORT luciferase vector to generate mutant PTEN plasmid. In the next step, wild‐type and mutant luciferase vectors were co‐transfected into KCL22 and K562 cells with LINC00470, and the luciferase activity of transfected cells was detected after 48 hours of transfection using a Wallac 1420 luminometer (Perkin Elmer) in conjunction with a Bright Glo luciferase reporter assay kit based on the directions provided by the assay kit manufacturer on the product manual.

Luciferase assays were carried out as previously described [8 (link)]. Briefly, complementary oligonucleotides corresponding to 54 nucleotides surrounding the miRNA binding site were annealed before being cloned into the SpeI and HindIII sites of pMIR-REPORT Luciferase (Ambion). A list of all oligonucleotides used is available in Table S6 in Additional file 1. All constructs were verified by capillary sequencing. HEK293T cells were co-transfected with 50 ng of a pMIR-REPORT Luciferase construct, 50 ng of pMIR-REPORT β-galactosidase (Ambion) and 10 nM of the appropriate mirVana miRNA mimic (Ambion/Life Technologies). After transfection, cells were incubated for 48 hours prior to harvesting. Luciferase activity was assayed using the Luciferase Assay System (Promega, Sydney, NSW, Australia), and detected on a Wallac 1420 luminometer (Perkin Elmer, Waltham, MA, USA). Transfection efficiency was assessed by β-galactosidase activity, using the β-Galactosidase Enzyme Assay System (Promega, Sydney, NSW, Australia), and detected on a PowerWave XS spectrophotometer (BioTek, Winooski, VT, USA).