Computational protocol: Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in Microglia: Stronger Effects of the A53T Mutant

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Protocol publication

[…] Total RNA was isolated from microglia using the innuPREP RNA Kits (Westburg, The Netherlands) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from RNA samples using the ImProm-II Reverse Transcription System (Promega, The Netherlands). PCR analyses were performed on a Bio-Rad iCycler (iQ5 Real-Time PCR Detection System, Bio-Rad) using iQTM SYBR Green Supermix (Promega). Primer sequences () were designed using the Beacon Designer Software (Bio-Rad). Gene expression was analyzed using the comparative threshold cycle (Ct) method. The target gene was normalized to the endogenous reference gene, Rpl27 (a housekeeping gene coding for a ribosomal protein). The mRNA expression fold change was calculated using the expression 2-ddCt, where ddCt = (Ct, target−Ct, Rpl27)treated sample−(Ct, target−Ct, Rpl27)control sample. [...] Data are represented as mean ± standard error of the mean (SEM) from at least three independent experiments. As normality could not be assessed on such samples, multiple group comparisons were made using a nonparametric analysis of variance (Kruskal-Wallis test) followed by pairwise comparisons with Dunn’s correction. All statistical analyses were performed using GraphPad Prism software and differences with p values less than 0.05 were considered significant.For PCR arrays, the HTqPCR package of bioconductor (R packages dedicated to biology) was used to complete the analysis. One of the HTqPCR data analysis allowed to tag Ct value as « OK », « undetermined » (Ct value above 35) or « unreliable » (Ct value outside the expected distribution, computed by HTqPCR) for each sample. Samples with less than 75% of value tagged as « OK » were removed from the dataset. Four normalization strategies (quantile, rank invariant, rank invariant scaled, and geometric mean) were applied to the data. Visual inspection of the normalization result through boxplot and comparison of two dispersion measures (coefficient of variation and standard deviation) lead to choose the quantile method to normalize data. The differentially expressed genes (DEG) list was generated using the Limma package. The DEG were selected when adjusted p value (Benjamini-Hochberg correction) < 0.05 and fold changes ≥ 2 criteria have been applied. […]

Pipeline specifications

Software tools Beacon Designer, HTqPCR, limma
Application qPCR
Diseases Parkinson Disease