Powerscript reverse transcriptase

Each total RNA sample from the gill and mantle was extracted with RNA-Bee™ (Tel-Test, Friendwood, TX, USA) according to the manufacturer's instructions. The RNA pellet was dissolved in 50 μL DEPC-H₂O and treated with the RNA clean-up protocol from the RNAspin Mini RNA isolation kit (GE Health Care, Piscataway, NJ, USA) according to the manufacturer's instructions to eliminate genomic DNA contamination. RNA integrity was verified by 0.8% agarose gel electrophoresis. Extracted RNA samples were stored at −80°C after isolation. First-strand cDNA was synthesized by reverse transcribing 5 μg of the total RNA using 1 μL of Oligo (dT) (0.5 μg μL⁻¹) primer and 1 μL of PowerScript™ Reverse Transcriptase (Clontech) according to the manufacturer's instructions.

Total RNA was extracted from LCLs, treated or not with DEX, using the RNeasyPlus mini kit (Qiagen) and cDNA was obtained by PowerScript reverse transcriptase (Clontech). Quantitative PCR for gene expression were performed by TaqMan Gene Expression Assays using the TaqMan Gene Expression Master Mix (Life Technologies) and run on a 7500 Real-Time PCR System (Applied Biosystems). For native ATM transcript quantification, we used the Hs01112314_m1 assay ID (ThermoFisher Scientific) that spans between exons 48–49; HPRT1 was used as a housekeeping gene.
End-point PCRs were performed on the same cDNA samples to identify any alternative splicing driven by the alteration. Two pairs of specific primers (Supplementary Table S1) were designed to amplify the regions from the start of exon 48 to the end of exon 50 and from the start of exon 48 to the end of exon 63 (last exon, using NM000051.4 as reference sequence).

The mRNA was purified from the total RNA extracted from tilapia tissues with a commercial kit (Oligotex, Qiagen, Hilden, Germany). For cDNA synthesis, 0.36 μg of mRNA was reverse transcribed in a final volume of 20 μl containing 0.5 mM dNTPs, 2.5 μM oligo (dT)₁₈, 5 mM dithiothreitol, and 200 units PowerScript reverse transcriptase (Clontech, CA, USA) for 1.5 h at 42 °C, followed by a 15 min incubation at 70 °C. For PCR amplification, 2 μl cDNA was used as template in a 50 μl final reaction volume containing 0.25 mM dNTP, 2.5 units EX-Taq polymerase (Takara, Shiga, Japan), and 0.2 μM of each primer. GenBank accession numbers of the sequences for primer sets were used as follows: ecac, GenBank BankIt Submission ID:1884659; pmca2, AAK15034; ncx1, AY283779; gadph, FN673690.
The primer sets used for Reverse transcription-PCR analysis were as follows: ecac (342 bp fragment), forward 5′-AGAGGATGAAAAGGAAACGG-3′, reverse 5′-ATGGCATAATACTGCGGAAA-3′; ncx1 (310 bp fragment), forward 5′-TGCCGTCTACCACTACACCC-3′, reverse 5′-GCAGCGACCTAAAATCCAAC-3′; pmca2 (693 bp fragment), forward 5′-AACAACCTGGTGCGTCA-3′, reverse 5′-GGGGTCCTCTATTCCGA-3′; gadph (415 bp fragment), forward 5′-AATACGACCCCTCCTCCAT-3′, reverse 5′-TACCCCAGCACTCCTTTCA-3′. The amplicons were all sequenced to ensure that the PCR products were the desired gene fragments.