Computational protocol: Altered 2-thiouridylation impairs mitochondrial translation in reversible infantile respiratory chain deficiency

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Protocol publication

[…] Isolation of RNA from both cells and tissues was carried out using Trizol® (Invitrogen) following the manufacturers recommendations. We performed northern blotting on APM containing gels, essentially using the method described previously (). This is the standard method to separate thiolated and non-thiolated tRNA species (). Following transfer to GeneScreen Plus membrane (Perkin Elmer), the presence of tRNA species was detected using 32P-labelled PCR products as described previously (). The probes for human mt-tRNAGlu, mt-tRNALys, mt-tRNAGln, the cytoplasmic tRNALys and 5S RNA were generated using primers listed in the Supplementary material. Quantification of the radioactive signal was performed with imageJ software. [...] The statistical package SigmaPlot 11.0 was used to perform all the statistics. Data are presented as mean ± standard deviation. The normal distribution was checked by the Kolmogorov–Smirnov test and ANOVA tests were used to compare parameters. The Holm–Sidak method was used for pairwise multiple comparison procedures. A P-value of <0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Mitochondrial Encephalomyopathies, Mitochondrial Diseases
Chemicals Cysteine