Effects of H2O2 on M. aeruginosa Circadian Rhythms Figure 8. Growth of M. aeruginosa that was cultured with H2O2 in the first 24 h. The shaded areas correspond to the dark period. Symbols represent mean 6 SEM of triplicate cultures. The open circle corresponds to the control treatment; the filled circle corresponds to the L0 treatment; the filled square corresponds to the D0 treatment. doi:10.1371/journal.pone.0033347.g008 fluorescent lights (4000 lux) for a daily 12-h-12-h light-dark cycle (LD cycle). The cell density of culture was monitored spectropho- tometrically at 685 nm (OD685). The regression equation between the density of cyanobacteria (Y6105/mL) and OD685 (X) was established as Y = 34.11X+0.73 (R2 = 0.99). When the culture reached the middle of the exponential growth phase (approximate 3.76105/mL), H2O2 (40 mM) was added to the culture at either the 0-h light point (termed L0 treatment) or the 0-h dark point (termed D0 treatment). In the LD cycle, the concentration of H2O2 was decided based on our previous report [10]. Sample collection began after 4 h of treatment and was performed every 4 h (up to 24 h). RNA extraction, reverse transcription and real-time PCR analysis Culture samples (30 mL) were collected by centrifugation for RNA isolation at each time point. The cell pellet for RNA isolation was quickly frozen in liquid nitrogen until RNA extraction. Total RNA was extracted using the RNAiso kit (TaKaRa Company, Dalian, China) following the manufacturer’s instructions. 500 ng RNA was reverse transcripted into cDNA according to the reverse transcriptase kit (Toyobo, Tokyo, Japan). Real-time PCR was carried out using an Eppendorf MasterCyclerH ep RealPlex4 (Wesseling-Berzdorf, Germany). A reaction mixture for each PCR run was prepared with the SYBRH Green real-time PCR Master Mix (Toyobo, Tokyo, Japan). Final concentrations in a total volume of 10 mL were: 5 mL of 26Master Mix (including SYBRH Green I, dNTPs, Mg2+, Taq DNA Polymerase, 16ROX), 0.4 mL of 10 mM each of specific sense and anti-sense primers, and 1 mL of cDNA, 3.2 mL of ddH2O. The following PCR protocol was used with two steps as our previous report [10]: one denaturation step at 95uC for 1 min and 40 cycles of 95uC for 15 s, followed by 60uC for 1 min. 16Sr DNA was used as a housekeeping gene to normalize the expression profiles. Four clock genes (kaiA, kaiB, kaiC and sasA) and six circadian genes (psaB, psbD1, rbcL, mcyA, mcyD and mcyH) were selected and the primer pairs for each are listed in Table S1. For verify the efficiency of amplification determined for Figure 9. The intracellular microcystin content of M. aeruginosa under light and dark conditions after H2O2 treatment. The shaded areas correspond to the dark period. The symbols represent the mean 6 SEM of triplicate cultures. The filled circle corresponds to the L0 treatment; the open circle corresponds to the control treatment; the filled square corresponds to the D0 treatment. doi:10.1371/journal.pone.0033347.g009 Figure 10. The change of microcystin content in water after oxidation by H2O2 for various time periods. doi:10.1371/journal.pone.0033347.g010 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33347