Targets of the small regulatory RNA DapZ. Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

To determine the targets of the small regulatory RNA DapZ in S. Typhimurium, we looked at the effect of a short pulse of DapZ over-expression on the Salmonella transcriptome, as well as a DapZ variant that lacks the GcvB-like R1 region. To achieve over-expression, the wild-type DapZ and its variant were cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 2 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674-1688. Overall design: We used whole-genome S. typhimurium microarrays to determine relative mRNA expression changes, comparing the mRNA profiles. Microarrays used in this study were produced by in-situ synthesis as 8x15k multipack format from Agilent Technologies. Each microarray comprises 13268 60-mer S. typhimurium strain SL1344 specific oligonucleotides supplemented with 319 60-mer S. enterica subsp. serovar Typhimurium 14028S specific oligonucleotides, 360 60-mer S. typhimurium LT2 specific oligonucleotides and 360 60-mer oligonucleotides specific for 149 Salmonella sRNAs. The experimental design involves the use of Salmonella enterica serovar Typhimurium genomic DNA as the co-hybridized control for one channel on all microarrays. Two independent biological eperiments were analyzed.

为鉴定鼠伤寒沙门氏菌中小调控RNA（small regulatory RNA, sRNA）DapZ的靶标，我们检测了短时脉冲式过表达DapZ对沙门氏菌转录组的影响，并同步考察了缺失GcvB样R1区域的DapZ突变体的效应。
为实现过表达，我们将野生型DapZ及其突变体克隆至pBAD质粒中，并用0.2%的L-阿拉伯糖诱导10分钟。随后我们提取总RNA用于转录组分析。以仅携带空pBAD质粒的菌株作为阴性对照，该对照同样经L-阿拉伯糖诱导。本实验设置2次生物学重复。该sRNA靶标鉴定策略已见于Papenfort等人发表于《Molecular Microbiology》（2006）第62卷第6期，第1674-1688页的研究成果。

整体实验设计：我们采用全基因组鼠伤寒沙门氏菌微阵列，通过对比不同样本的mRNA表达谱，检测相对mRNA表达变化。本研究使用的微阵列由安捷伦科技（Agilent Technologies）以原位合成法制备，采用8×15k多包格式。每张微阵列包含13268条针对鼠伤寒沙门氏菌SL1344菌株的60聚体寡核苷酸探针，此外还补充了319条针对肠炎沙门氏菌亚种鼠伤寒血清型14028S菌株的60聚体寡核苷酸探针、360条针对鼠伤寒沙门氏菌LT2菌株的60聚体寡核苷酸探针，以及149条针对沙门氏菌sRNA的60聚体寡核苷酸探针。
本实验设计规定，所有微阵列的一个通道均以肠炎沙门氏菌鼠伤寒血清型的基因组DNA作为共杂交对照。本研究共分析了2次独立的生物学实验。