Computational protocol: Reprimo tissue-specific expression pattern is conserved between zebrafish and human

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Protocol publication

[…] In order to identify potential differentiating amino acid substitutions to distinguish between RPRM, RPRML and RPRM3, we annotated RPRM genes in human, mouse and zebrafish. Amino acid sequences were aligned using the L-INS-i strategy from MAFFT v.7 []. Additionally, potential domains were predicted using the TMHMM method (http://www.cbs.dtu.dk/cgi-bin/nph-sw_request?tmhmm), as implemented in Geneius Software. [...] All real-time PCR experiments were performed using a Stratagene Mx3000P detector system (Agilent Technologies) with optic tubes (SSI Innovations for Life Science). Amplification was achieved using Brilliant II SYBR® Green QPCR Master Mix (Agilent Technologies), equal amount of cDNA, and gene specific primers sets: rprma, fw: 5- AACCAAACGGACAGTGGCATCT-3’, rv: 5’- AAGACTACGGTGAGGGAAAGCA-3’; rprmb, fw: 5’- GGCTGCAACTTGCTGATTAAGTCC-3’, rv: 5’- GATGACCGCTTCCACATCCTTTGA-3’; rprml, fw: 5- ACGAGCGCAAACTGTTCGTTAC-3’, rv: 5- TCA TGAGGTTGCAGCCGAGAA A-3’; rprm3, fw: 5’- TGTTTCTCACGGACTACTGAACC-3’, rv: 5’- TAATACGACTCACTATAGGGTGCAGCGATCATAATAATTTCC-3’. Following completion of each real-time PCR reaction, a dissociation step was added and melt curve analysis was performed to validate the specificity of PCR amplicons. Data were processed by MxPro qPCR Software 4.10. rprm mRNA expression levels were normalized against an endogenous control gene previously validated as suitable reference gene for developmental studies in zebrafish []: actb1: fw:5’ -CGAGCAGGAGATGGGAACC-3’, rv: 5’-CAACGGAAACGCTCATTGC- 3’. Relative expression was calculated by ΔCT method against the housekeeping gene actb1. Expression plots were elaborated using R statistical programming language package ggplot2 and represented as average relative expression +/- SEM or relative expression boxplots, for developmental and adult expression, respectively. […]

Pipeline specifications

Software tools MAFFT, TMHMM, Ggplot2
Applications Miscellaneous, Membrane protein analysis
Organisms Danio rerio, Homo sapiens