Improm 2 reverse transcriptase 2

For adult expression analysis of rprma, rprmb and rprml, 4 adult TAB5 wild-type male zebrafish were euthanized for organ extraction, according to the protocol by Gupta & Mullins (10.3791/1717). Zebrafish brain and bowel were harvested and homogenized in TRI-Reagent^® (Sigma-Aldrich). Total RNA was collected separately for each sample, according to manufacturer’s instructions. A 2μl aliquot of each extract was used for RNA quantification, quality assessment and concentration by spectrophotometry (BioSpectrometer, (Eppendorf)).
cDNA was synthesized from total RNA (1 μg; 20 μl final reaction volume) with oligo (dT) priming using ImProm-II Reverse transcriptase II (PROMEGA) according to manufacturer's instructions.

For developmental expression analysis of rprma, rprmb, rprml and rprm3; embryos were collected after timed intervals of 0.2, 6, 12, 24, 31, 48, 72 and 96 hours post-fertilization (hpf), quick-frozen on liquid nitrogen, and stored at -70°C until analysis (3 independent embryo pools, 50 embryos per pool, per time point from the same spawning group). Whole embryos were homogenized in Lysis Buffer (Thermo Scientific) and total RNA was extracted with GeneJet RNA Purification kit (Thermo Scientific) according to manufacturer's instructions. Embryo actin, beta 1 (actb1) was used for housekeeping gene expression analysis and gene of interest normalization. An aliquot (2μl) of each extract was used for RNA quantification, quality assessment and concentration by spectrophotometry (BioSpectrometer, (Eppendorf)). RNA with a 260/280 ratio between 1.6–2.0 was considered optimal in this study. Each RNA extract was performed in triplicate and an average value was determined. cDNA was synthesized from total RNA (1 μg; 20 μl final reaction volume) with oligo (dT) priming using ImProm-II Reverse transcriptase II (PROMEGA) according to manufacturer's instructions.

For cDNA synthesis, total RNA was collected from pools of 50 rprml MO-injected and 50 uninjected control embryos at 48 hpf using E.Z.N.A total RNA kit I (OMEGA), according to manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA with oligo (dT) using ImProm-II Reverse transcriptase II (PROMEGA) following manufacturer’s guidelines.