Bioluminescence assays

Luciferase activity was assayed by growing C. reinhardtii cultures with miRNA-overexpressing constructs or control constructs in transparent 96-well-plates containing 200 μL liquid TAP per well under constant illumination (approximately 25 μE·m⁻²·s⁻¹) to a final cell density of 3–6 × 10⁶ cells·mL⁻¹ after three days. After sampling, cells were centrifuged at 10,000 g for 1 min at 25 °C. The cell pellet was resuspended in 1 mL phosphate buffered saline (PBS; pH = 7.4) and centrifuged at 10,000 g for 1 min at 25 °C to remove the remaining TAP medium. The cells were resuspended in 50 μL lysis buffer following incubation at room temperature for 15 min. The cell lysate was allowed to undergo 2 or 3 freeze-thaw cycles to ensure complete lysis of cells. After thawing, 50 μL of the cell lysate was transferred to 96-well black plates, and 50 μL of the assay buffer was added to each well. Bioluminescence was immediately assayed using a multi-mode microplate reader (Filter Max F5; Molecular Devices). The background was normalized by measuring bioluminescence of wells containing only buffer and those containing buffer with cells having the pHK225-vector.