strain: AB tissue: all tissue anterior to gills treatment: KD time: 15DPF
Treatment protocol
In accordance with current guidelines for morpholino use in zebrafish, the use of morpholino-modified antisense oligonucleotides to knockdown (KD) grb10a was validated in several ways. Morpholinos targeting two distinct sites should yield a similar phenotype, which should display a dose-response effect and be rescuable by RNA co-injection, and a suitable control morpholino with no phenotypic effect should be used to account for microinjection technique. dpf or four (e4i4) were designed by and obtained from Gene Tools, LLC (Philomath, OR, USA) along with a standard control (SC) oligonucleotide targeting human β-globin. Concentrations of ingredients were consistent with standard practice. Phenol red and nCerulean (nuclear-targeting blue fluorescent protein) mRNA were included in each solution to allow monitoring of successful injection. A P-97 Flaming/Brown Micropipette Puller (Sutter Instruments, Novato, CA, USA) was used to generate microinjection needles from 1 mm OD glass capillaries, which were backloaded with 2 μl of injection solution and mounted on a needle holder held in a micromanipulator attached to a PLI-100A Picoliter Injector (Warner Instruments, Hamden, CT, USA). Embryos were injected once, as per established method, into the yolk directly below the cell mass. Embryos were injected before the four-cell stage and were screened for fluorescence at 48 hours post fertilisation (hpf) to ensure constitutive and even uptake of the injection material. Non-uniformly or weakly-fluorescent embryos were removed.
Growth protocol
AB zebrafish were maintained under standard conditions (≈28 °C; 14 h light/10 h dark cycle; stocking density < 5 fish per litre) within the Biological Services Unit of The University of Manchester. Embryos were collected and raised in egg water (Instant Ocean salt 60 µg/mL) up to 5 days post-fertilisation (dpf) and transferred to the main aquarium system. Young fry were fed powdered food and rotifers, while older fry and adults were fed powdered food and brine shrimp (ZM Fish Food and Equipment, Winchester, UK). All regulated procedures received ethical approval from the Institution’s ethical review board and were performed under a Home Office Licence (PPL P005EFE9F9 to HAS).
Extracted molecule
total RNA
Extraction protocol
Total RNA, Qiagen Rneasy
Label
biotin
Label protocol
GeneChip WT PLUS Reagent Kit for Affymetrix Zebgene 1.0st
Hybridization protocol
RGT KIT,HYB WASH STAIN for Affymetrix Zebgene 1.0st
Scan protocol
Arrays were scanned on an Affymetrix GeneChip scanner and assessed for quality against internal and hybridization controls.
Data processing
Gene level expression was determined and processing and normalization of gene expression data were performed using a Robust Multi-array Average background correction with quantile normalization. Analysis performed in Qlucore Omics Explorer. Log2 normalised and scaled (mean of 0, standard deviation of1) RMA values from Qlucore Omics Explorer. Gene level summary of array data.
