A Multisampling Reporter System for Monitoring MicroRNA Activity in the Same Population of Cells
Figure 3
Increase in miR-16 activity after EGCG treatment of HepG2 cells. HepG2 cells were grown in 5 mL of DMEM in 6cm Petri dishes to 70% confluency. (a) The cells were cotransfected for 6 hours with 4 g of pMLuc or pMLuc-UTR and 4 g of pSEAP2-Control, then 5 L of 100 mM of EGCG (dissolved in DMSO) or DMSO was added. The medium was replaced with fresh medium at 24 and 36 hours and an aliquot of medium was withdrawn 2 hours later and assayed for MLuc and SEAP. The fold repression is defined as the MLuc (from pMLuc)/SEAP ratio divided by the MLuc (from pMLuc-UTR)/SEAP ratio. (b) The cells were treated with or without EGCG for 24 or 36 hours, then total RNA was extracted and subjected to qRT-PCR to detect levels of miR-16 and U6 small nuclear RNA (see Section 2 for details). The data presented are the mean ± SD of triplicates;
