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. 2011 Jan 25;2(1):e00340-10. doi: 10.1128/mBio.00340-10

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Copyright © 2011 Chen et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

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FIG 1 — Q-RT-PCR validation of a select set of predicted sRNAs in Clostridium acetobutylicum and three Clostridium botulinum strains. Cycle threshold (C_T) values are shown for genes in four groups: positive-control genes, predicted sRNAs, low-expression genes, and negative-control genes. C_T values are averages of 5 or 6 replicates (error bars represent standard deviations). The numbers shown above the symbols (1 to 47) represent specific genes as listed below; the gene name is given first, and the gene number is shown in parentheses after the gene name. (A) Q-RT-PCR test of sRNA expression in C. acetobutylicum using pool CAC1 as described in Text S1 in the supplemental material. Positive-control genes (genes 1 to 6) include the 16S rRNA and genes known to be expressed well on the basis of microarray data (34): 16S (1), 6S (2), CAC0681 (3), CAC2957 (4), CAC1322 (5), and CAC2139 (6). Low- or no-expression genes (under normal culture conditions) (genes 37 to 42) based on microarray data (34) include the following: CAC3313 (37), CAP0060 (38), CAC0428 (39), CAC1094 (40), CAC2614 (41), and CAC2179 (42). The negative controls (genes 43 to 47) include the following intergenic region (IR) sequences (Ig stands for intergenic): IgCAC_1350 (43), IgCAC_232 (44), IgCAC_2999 (45), IgCAC_2630 (46), and IgCAC_2996 (47). The predicted sRNAs (genes 7 to 36) include the following: sCA-P60 (7), sCAC1449 (8), sCAC3821 (9), sCAC137 (10), sCA-P189 (11), sCAC3283 (12), sCAC1645 (13), sCAC646 (14), sCAC1132 (15), sCAC1582 (16), sCAC1760 (17), sCAC2795 (18), sCAC3723 (19), sCAC903 (20), sCAC3340 (21), sCAC3825 (22), sCAC975.1 (23), sCAC1315 (24), sCA-P18 (25), sCAC610 (26), sCAC2819.1 (27), sCAC1313 (28), sCA-P105 (29), sCA-P18.1 (30), sCAC3039 (31), sCAC500 (32), sCAC1594 (33), sCAC2709 (34), sCAC3850 (35), and sCAC349 (36). Notice that the values of the genes expressed well (genes 4 to 6) and the low-expression genes (genes 37 to 42) overlap. Thus, three zones of approximate expression are indicated: expressed (C_T ≤ 21), lowly expressed if at all (21 < C_T < 24), and probably not expressed (C_T ≥ 24). The C_T value of 21 was chosen as just below the C_T value of the lowest low-expression control genes; the C_T value of 24 was chosen based on the subsequent finding (Fig. 3) that sCAC610 represented by gene 26 is highly expressed. (B) Q-RT-PCR test of sRNA expression in C. botulinum A3 Loch Maree,C. botulinum A ATCC 3502, and C. botulinum A Hall using pool CLK, pool CBO, and pool CLC, respectively, as described in Text S2 in the supplemental material . The positive controls include 23S rRNA (genes 48 to 50) and 16S rRNA (genes 51 to 53) in the three C. botulinum strains. The negative controls (genes 75 to 78) include the following intergenic region sequences: IgCLC_2904 (75), IgCLC_2422 (76), IgCLC_2026 (77), and IgCLC_2348 (78). The predicted sRNAs (genes 54 to 74) are sCLK_200 (54), sCLK_3269 (55), sCLK_3642 (56), sCBO3039 (57), sCLC_2889 (58), sCLK_3105 (59), sCLK_3427 (60), sCBO2696 (61), sCLK_3557 (62), sCBO3480 (63), sCBO1976 (64), sCLK_2040 (65), sCLK_2759 (66), sCLC_3353 (67), sCLC_1905 (68), sCLC_2101 (69), sCBO2173 (70), sCLC_3476 (71), sCBO3602 (72), sCLK_1206 (73), and sCLK_3693 (74).