Nanodrop 2000c spectrophotometer

Total RNA was extracted using an RNA prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China). First-strand cDNA was synthesized using a reverse transcription system kit (Nanjing Vazyme Biotech). The full coding sequence of the non-membrane region of EgHCDH was amplified using the primers 5′- CGG GAT CCA TGT CAG CCG GTG CTG G-3′ (BamHI) and 5′-GAC GTC GAC TCA CTG TTT TTC CTT GAC AAT GCG C-3′ (SalI). Amplification reactions were performed using the following cycling conditions: pre-denaturation at 95 °C, 5 min; then denaturation at 95 °C/30 s, 62 °C/30 s, 72 °C/1 min; with a final extension at 72 °C, 5 min. Through sequencing, digestion and ligation, EgHCDH was ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Protein expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 6 h. The rEgHCDH protein was purified using Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA), with the the purity of the final product determined by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of the purified protein were determined using a NanoDrop 2000c spectrophotometer (Bio-Rad).

pGEM-T easy plasmids containing the full coding regions of the zebrafish mcr1 and mcr4 genes [4 (link)] were linearized with SalI or ApaI and used to prepare antisense and sense riboprobes by in vitro transcription using T7 or SP6 RNA polymerase (Promega, Spain), respectively and digoxigenin (DIG)-labelled UTPs (Roche Diagnostics GmbH). Synthetized probes were treated with RQ1-DNAse-RNAse free (Promega, Spain) for 15 min at 37 °C to remove the DNA template. Ultimately, the probes were purified using Micro Bio-Spin Chromatography Columns (BioRad, Spain) and quantified in a Nanodrop 2000c spectrophotometer.