Electronic images were auto-leveled and relevant lanes were placed side-by-side using Adobe Photoshop CS3. Micro-array analysis assisted transcript mapping To complement the RT-PCR and Northern analyses, we hybridized Cy3-labeled cDNA synthesized from total RNA isolated from P. knackmussii B13 cultures during exponential growth on 3-chlorobenzoate and during the following stationary phase, to custom-designed semi-tiling microarrays for ICEclc. The semi-tiling array contained a 50-mer probe at approximately every 200 bases over the whole length of ICEclc and for both strands, each in sixfold replicate on the array. We expected that a semi-tiling array format would permit us to map the position of ICEclc transcripts in a complementary
way to the conventional molecular analysis, which would help to reinforce the conclusions drawn on the transcriptional organization of the ICEclc core. Figure 4 shows an overlay LY2835219 in vivo of the core gene organization and RT-PCR plus Northern derived transcriptional organization with the average micro-array hybridization signals per probe on the plus- and the minus-strand of the ICEclc core region, whilst Table 1 summarizes the transcript details across all three methods. Very strikingly, most
of the predicted transcripts follow a clear 5′-3′ decrease in signal intensity, the slopes of which were different for each transcript region (see, for example, the region for the long transcript proposed between position 82,000 and 68,000). We think the 5′-3′ decrease in intensity may partially be caused by the fact that more transcripts are formed near the Evofosfamide datasheet transcription start, which perhaps are incompletely finished, or by preferential 3′-end degradation. This effect has been noted by others using tiling
approaches for transcript determination . Different slopes may be the result of varying mRNA stability and processing speed. Figure 4 Transcriptome of the ICE clc core region. Shown is a compilation of micro-array hybridizations with minus- (top image) and plus-strand located probes (bottom image), both for exponential (yellow Ruxolitinib solubility dmso squares and blue circles) and stationary phase cultures (dark squares and pink circles). Data points are mean hybridization signals (on log2-scale) from six replicate probes per array, averaged over three replicate arrays.). X-axes, position numbering on ICEclc. Middle SB-3CT part, representation of the gene locations in the ICEclc core region (block arrows), and the size and position of the transcripts concluded from RT-PCR and Northerns (Figure 1-3). Table 1 Summary of ICEclc core transcripts. Transcript Stranda Size on Northernb RT-PCRc Promoterd Log2 Stat-Expo Ratioe intB13 + 2.5 + 102,729 (Pcirc) 3.1 ± 1.0 ORF50240 – ND (1.8) ND 1.6 ± 0.6 52324-53196 + 1.5 (1.2) + 51,218 -1.2 ± 0.4 53587-58432 – 4.7 (5.3) + 58,771 4.2 ± 1.4 59110-62755 – 3.5 (4.0) + 63,191 2.6 ± 1.3 63176-66202 – 3.5 (3.4) + 66,976 2.3 ± 1.7 66625-67231 – ND (1.0) + 67,610 5.8 ± 2.2 67800 + 2.