The differential expression of Mif-Csn-Nf-kB axis LPS-responsive genes was studied by qRT-PCR using the SYBR-Green method and the specific sets of primers listed in File S1 . qRT-PCR analysis was performed using an Applied Biosystems 7500 Real-time PCR system [61 (link),62 (link)]. Differential expression was determined in a 25 μL PCR mixture containing 2 μL of cDNA converted from 250 ng of total RNA, 300 nM primer (forward and reverse), and 12.5 μL of Power SYBR-Green PCR MasterMix (Applied Biosystems, Waltham, MA, USA).
Amplification specificity was tested using a real-time PCR melting analysis. To obtain sample quantification, the 2−ΔΔCt method was used, and the relative changes in gene expression were analyzed as described in the Applied Biosystems Use Bulletin N. 2 (P/N 4303859).
The transcript levels from different tissues were normalized to that of actin to compensate for variations in the amount of RNA input. Relative expression was determined by dividing the normalized value of the target gene in each tissue by the normalized value obtained from the untreated tissue. To examine the time course of the response, 4 LPS-treated ascidian replicates (n = 4) were examined at incremental post-inoculation time points (1, 2, 4, 8, 12, 24, and 48 h). Four untreated (naïve) ascidian replicates (n = 4) were used as controls. A heatmap was generated to visualize the results indicating the genes differentially expressed between the exposed samples and controls (LPS exposure times were 1 h, 2 h, 4 h, 24 h, and 48 h). The Minitab 17 statistical software was used for the qRT-PCR data analysis. Statistical differences were estimated by a one-way ANOVA, and the significance of differences among groups was determined by Tukey’s t-test. The level of significance was set at a p-value ≤ 0.05. The data are presented as the means ± SD (n = 4).
The heatmap was produced using a heatmapping tool (https://www.heatmapper.ca accessed on 1 June 2022). Complete linkage clustering was applied, and Pearson correlation was used as the method of distance measurement. Additionally, a z-score was calculated, a measure that describes a value’s relationship to the mean of a group of values. The z-score was measured in terms of standard deviations from the mean [63 (link)].
Amplification specificity was tested using a real-time PCR melting analysis. To obtain sample quantification, the 2−ΔΔCt method was used, and the relative changes in gene expression were analyzed as described in the Applied Biosystems Use Bulletin N. 2 (P/N 4303859).
The transcript levels from different tissues were normalized to that of actin to compensate for variations in the amount of RNA input. Relative expression was determined by dividing the normalized value of the target gene in each tissue by the normalized value obtained from the untreated tissue. To examine the time course of the response, 4 LPS-treated ascidian replicates (n = 4) were examined at incremental post-inoculation time points (1, 2, 4, 8, 12, 24, and 48 h). Four untreated (naïve) ascidian replicates (n = 4) were used as controls. A heatmap was generated to visualize the results indicating the genes differentially expressed between the exposed samples and controls (LPS exposure times were 1 h, 2 h, 4 h, 24 h, and 48 h). The Minitab 17 statistical software was used for the qRT-PCR data analysis. Statistical differences were estimated by a one-way ANOVA, and the significance of differences among groups was determined by Tukey’s t-test. The level of significance was set at a p-value ≤ 0.05. The data are presented as the means ± SD (n = 4).
The heatmap was produced using a heatmapping tool (
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