Computational protocol: Computational inference and experimental validation of the nitrogen assimilation regulatory network in cyanobacterium Synechococcus sp. WH 8102

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Protocol publication

[…] Total RNA from exponential phase cells was extracted using a Trizol-based method and purified using a Qiagen RNeasy kit following manufacturer's instructions. An indirect labeling method is used to label cDNA, where cDNA is synthesized in the presence of a nucleoside triphosphate analog containing a reactive aminoallyl group to which the fluorescent dye molecule is coupled. Control and treated samples are labeled with Cy3 and Cy5, respectively, pooled and hybridized to the same array. In addition, reverse labeling was routinely performed to reduce dye-specific biases in signal intensity. Slides were hybridized with labeled cDNA and then scanned and analyzed using TIGR's SPOTFINDER, MADAM and MIDAS software (). RNAs from five nitrate grown cultures and four ammonium grown cultures were used. Thirteen different hybridizations were carried out, of which six used different RNA pools and seven were replicates of these six experiments, either dye swapping experiments or direct replicates. Statistical analyses were carried out using the Significance Analysis of Microarrays (SAM) software package (), and the thirteen hybridizations were treated as independent experiments. SAM orders the genes by using a modified t statistic called relative difference based on the ratio of change in gene expression to standard deviation in the data for that gene. It declares a gene to be up- or down-regulated if the difference (D-value) between the observed relative difference and expected relative difference is above (D-value > 0) or below (D-value < 0) the global cutoff point, respectively (). This procedure allows estimation of the median of false discovery rates. In this study we selected the cutoff points to control this median to be at most 5%. We call that a gene was down- or up-regulated by ammonium if it has a negative or positive D-value beyond the selected cutoffs, respectively. […]

Pipeline specifications

Software tools Spotfinder, SAM
Application Gene expression microarray analysis
Organisms Synechococcus elongatus PCC 6301
Chemicals Quaternary Ammonium Compounds, Nitrogen