Comparison of microarray and qRT-PCR-based quantification of transcripts in a diurnal rhythm.

<p>Changes in organelle (chloroplast and mitochondrial) transcript levels between time points L2 and D2 in a wild-type strain of <i>C. reinhardtii</i> (CC-124) were determined by microarray and by qRT-PCR. The resulting fold-changes correlate with an R² value of 0.66. It is lower than 1.0, mainly because the microarray produces less accurate results for lowly expressed genes. All data points represent the average of three independent experiments.</p

k-means clustering of diurnal qRT-PCR data for chloroplast transcripts (left) and a subset of nuclear transcripts (right).

<p>The averaged data for six biological replicates were grouped into three clusters with the Pearson correlation as the distance metric. Gene profiles are colored according to the gene product’s function. Green: photosynthesis-related genes, blue: ribosome-related genes, red: plastid-encoded RNA polymerase genes, grey: miscellaneous genes. Clustering and visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p

Diurnal transcriptomics on bioreactor cultures of <i>Chlamydomonas</i>.

<p>The effect of 12 h light/12 h dark cycles on organelle (and selected nuclear) transcripts was analyzed by qRT-PCR. <i>C. reinhardtii</i> cells were grown in six independent bioreactor runs (run numbers 1, 2, 3, 15, 16, 20) and harvested at the time points indicated. Data obtained for plastome transcripts is shown in the upper portion of the heatmap, chondriome transcripts in the central region, and nuclear transcripts in the lower region. Within each genome, transcripts are listed alphabetically. Data are normalized to housekeeping transcripts and then to the average across all samples for that gene. Red boxes indicate up-regulation, green boxes: down-regulation, grey boxes: no data. Visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p

Confirmation of the diurnal expression patterns observed in the pRT-PCR experiments by northern blot analyses.

<p>To avoid variation from RNA loading, each membrane was hybridized and stripped multiple times. The order of probing corresponds to the vertical order of the images (and the increasing signal intensity known to be obtained with the different probes). The ethidium bromide fluorescence (EtBr) remaining on the membrane after blotting is shown as a loading control.</p

Behavior of functionally related chloroplast transcripts in diurnal conditions.

<p>The average peak time of all transcripts belonging to the three functional classes (red: plastid-encoded RNA polymerase genes, PEP; blue: ribosome-related genes; green: photosynthesis-related genes) is shown for each bioreactor experiment. The intensity of the circle represents the number of transcripts peaking at the time, and the black x represents the average of all transcripts. The yellow bar represents the 12 h light period, flanked by dark periods (grey bars).</p

Key bioreactor parameters from a representative bioreactor run (R2) in diurnal conditions.

<p>Turbidity (navy blue) slightly decreased during the dark period and increased during the light. The rapid peak after dusk and rapid drop after dawn are technical artefacts of the turbidimeter. The peaks in the weight graph correspond to the sampling times, when the fermenter was disturbed. Dissolved oxygen concentration increases from ca. 80% to ca. 150% very rapidly in the light. The pH of the culture is controlled by titration of acid or base, temperature is controlled by dynamic cooling to maintain reasonably stable values for these parameters. Samples were taken every 4 h, these points are indicated and also visible as peaks in the weight data (purple). Yellow bars represents the 12 h light period (L), flanked by dark periods (D; grey bars).</p