Computational protocol: Deficient Vitamin E Uptake During Development Impairs Neural Tube Closure in Mice Lacking Lipoprotein Receptor SR-BI

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Protocol publication

[…] We analysed the dataset generated by Hannibal et al., which was produced by RNA sequencing of polyA+ RNA from isolated mouse E9.5 parietal TGC. The Single End Reads RNA raw sequence files (.fastq files) were extracted from GEO Accession Number GSE50585. We used the entries GSM1223565, GSM1223566, GSM1223567 and GSM1223568 to generate our data, which underwent quality control analysis using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Single End RNAseq reads were groomed using FASTQ groomer V1.04, and groomed reads were mapped to the mouse genome (mm10) using TopHat2 version 0.7. Aligned reads were counted using HTseq-count Version 1.0.0 to generate a digital expression matrix. To ensure that all of the sequences were processed consistently, all of the above steps were performed as part of a Galaxy workflow, which can be found at https://usegalaxy.org/u/laiumiunix/w/rnaseq.A list of all of the lipoprotein receptors was obtained from the Gene Ontology Consortium using the “Lipoprotein particle receptor activity” and “Regulation of plasma lipoprotein particle levels” search terms. The positive controls were marker genes of parietal TGC, whereas the negative controls were markers of TGC from a different lineage that does not give rise to parietal TGC. We considered a gene to be expressed by TGC when the mean read count of that gene was superior to the mean +3 SD of Tpbpa, the negative control with the highest count. This criterion set the threshold at 71 mean reads. [...] Total RNA was extracted from pools of 3 female embryos and individual parietal yolk sacs using the PureLink RNA Micro Kit (Invitrogen, CA), following the manufacturer’s instructions. The embryos used to make the pools came from 9 control litters and 6 vitamin E-supplemented litters. RNA integrity was evaluated using the Bioanalyser 2100 (Agilent, CA) with the Eukaryote Total RNA Nano assay (Agilent, CA). All samples had an RNA Integrity Number of 10. Purified RNA (500 ng) was used for retrotranscription with the iScript RT Supermix (Biorad, CA). The resulting cDNA was amplified by real time PCR with a StepOnePlus thermocycler (Applied Biosystems, CA) using the PowerUp SYBR Green master mix (Thermo, MA) and 100 nM of each primer. The primers, annealing temperatures and amplification efficiencies are listed in Supplementary Table . All primers were designed using NCBI’s Primer-BLAST. The amplification conditions were as follows: 5 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C, 15 seconds of annealing and 30 seconds at 72 °C. After every reaction, a melting curve was performed to ensure the amplification of a single product. The amplification efficiency of each pair of primers was determined by serial dilution of a mixture of the cDNAs. Then, the relative expression was calculated for each sample using the equation by Pfaffl (equation 1 in the reference) and the TATA-box binding protein (Tbp) as reference gene. [...] Sample sizes were calculated to achieve an 80% power of detecting a 2-fold change with α = 0.05. For supplementation with α-tocopherol after implantation, we sought an 80% power to detect a 95% reduction in the presence of NTD in SR-BI−/− embryos with α = 0.05.The assignment of pregnant dams to each treatment group was pseudo-randomized. Each day, the first female with a vaginal plug was assigned to the control group, the second one to one of the treatment groups, and so on. If only one female had a plug one day, the next day the order was reversed. The phenotypic assessments were performed blinded to the genotype of the embryo, but not to the treatment group. Biochemical and real time PCR experiments were performed blind to the genotype and the treatment group of the sample.Results are shown as scatter plots with a horizontal line indicating the mean (or median where indicated) for arithmetic data, mean ± SEM for lipoprotein profiles and geometric mean + error for exponential data obtained from real time PCR experiments. The error represents the uncertainty in estimating the relative expression and is computed using Taylor’s series relative to the control group. Therefore, error is reported only for the non-control groups.The statistical significance of the difference between proportions was evaluated with the Fisher’s exact test. Differences between arithmetical means were tested for significance using one-way ANOVA with a Tukey’s post-hoc test or two-way ANOVA with the Holms-Sidak post-test. If variances were different between groups, then a non-parametric test was used (Mann-Whitney for two group comparison and Kruskal-Wallis with Dunn’s post-test for multigroup comparison). The significance of the difference in gene expression was tested using the Pair-wise Fixed Reallocation Randomization test with the Relative Expression Software Tool Multiple Comparison Solution.All tests were two-sided, and results were considered significant at p ≤ 0.05. The statistically significant differences between groups are symbolized by asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). […]

Pipeline specifications

Software tools FastQC, Galaxy, TopHat, HTSeq, Primer-BLAST, REST
Applications RNA-seq analysis, qPCR
Organisms Mus musculus, Caenorhabditis elegans
Diseases Neural Tube Defects
Chemicals Cholesterol, Oxygen, Vitamin E