Computational protocol: Cnbp ameliorates Treacher Collins Syndrome craniofacial anomalies through a pathway that involves redox-responsive genes

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Protocol publication

[…] ROS abundance was detected in injected 48 hpf embryos by measuring the intracellular level of the oxidized form of acetyl ester of dichloro-dihydro-fluorescein diacetate, as described elsewhere., For western blotting, 24 hpf embryos were dechorionated and processed according to Link et al. SDS-PAGE was performed essentially according to Laemmli. Gels were electrotransferred to nitrocellulose membranes according to Towbin et al. Rabbit polyclonal anti-actin (I-19; sc-1616-R), mouse monoclonal anti-tubulin (TU-02; sc-8035) and rabbit polyclonal anti-p53 (FL-393; sc-6243) were from Santa Cruz Biotechnology. Rabbit polyclonal anti GFP (ab290) was from Abcam (Cambridge, UK). Cnbp anti-serum was raised in rabbit against the zebrafish full-length protein. Membranes were incubated with the appropriate primary and HRP-conjugated antibodies (1/7000 to 1/25000 dilution), washed and developed using chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific) and X-ray films (Amersham Hyperfilm ECL, GE Healthcare Science, Chicago, IL, USA).Relative protein concentrations were determined according to Sedmak and Grossberg, using bovine serum albumin as standard.Total RNA from 24 hpf embryos was obtained using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and following the manufacturer's instructions. Purified RNA was incubated with RQ1 DNAse (Promega, Madison, WI, USA) and oligo(dT) retrotranscribed with M-MLV reverse transcriptase (Promega). Quantification reactions were performed using three different RNA purifications from three independent microinjection experiments using an Eppendorf Realplex2 apparatus and SYBR green I (Invitrogen) chemistry. Each reaction tube (20 μl) consisted of 0.5 × SYBR Green I, 0.2 μM of each primer (), 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 U Platinum Taq DNA polymerase (Invitrogen) and 2 μl of template/negative controls. Ef1α and Rlp13a were used as endogenous controls for gene expression normalization. Relative gene expression values were calculated using qBase v 1.3.5. Statistical differences were analyzed by ANOVA and Student's t-tests. The validity of the RT-qPCR data was assured by following the MIQE guidelines.Ribosomal RNA transcription was estimated via RT-qPCR as elsewhere. Briefly, two pairs of primers () were designed against the 5′ETS and ITS1 regions of the unprocessed transcript (47S) based on Azuma et al. Total RNA from 256-cell stage and 24 hpf embryos were used in retro-transcription reactions using random primers. Actin and Rlp13a were used as endogenous controls for gene expression normalization.All the oligonucleotides used () were purchased from GenBiotech (http://www.genbiotech.com.ar/). Specific oligonucleotide primers for each gene under study were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and their specificity checked using MFE http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/. […]

Pipeline specifications

Software tools Primer-BLAST, MFEprimer
Application qPCR
Organisms Danio rerio, Homo sapiens, Mus musculus, Caenorhabditis elegans
Diseases Bone Diseases, Cleft Palate, Mandibulofacial Dysostosis, Craniofacial Abnormalities