Supermix iscript cdna synthesis kit

Trizol solution (Life Technologies 15596-026) was used to collect RNA from cell spheroids that were cultured for the referred periods. For larger, ULA spheroids, 16–24 spheroids were pooled for RNA collection, while several hundred of the smaller, AggreWell-formed spheroids were used. The collected RNA was later isolated through phenol-chloroform extraction following manufacturer’s instructions. Samples were digested through DNAse (New England Biolabs, M0303S) at 37°C for 30 min and cleaned using an RNeasy Mini Kit (Qiagen, 74104). RNA concentration was measured by UV spectroscopy and only samples with a ratio >1.8 were used for cDNA preparation. cDNA from isolated RNA was generated using the Supermix iScript cDNA synthesis kit (BioRad, 1708841) and mixed with pre-added primer pairs at a final concentration of 100 nM/primer, again per manufacturer’s instructions. Primer pairs for each gene were designed as previously shown using the NCBI’s Primer-BLAST with a target Tm of 60°C (see Supplementary Table 1 for primer pair sequences). Thermal cycling and measurement of amplification curves were executed through the CFX Connect Real-Time PCR Detection System (Bio-Rad) and mRNA expression was calculated relative to Hprt1 and control samples as indicated, with n ≥ 3 biological replicates, unless otherwise noted.

Total RNA was extracted from the liver and muscle tissues using TRIzol (Invitrogen, USA) following the manufacturer’s protocol. The concentration and quality of RNA were measured using ScanDrop 200 (Analytik Jena, Germany). cDNAs were synthesized from 1 µ g total RNA using the iScript Supermix cDNA Synthesis Kit (Bio-Rad, USA).
A fragment of GPx was amplified using degenerate primers by Choi et al.^{24 (link)} This fragment was used to design species-specific primers for rapid amplification of cDNA ends (RACE) by Primer3 program.^{25 (link)} To obtain full-length sequences of GPx gene for T tambroides, RACE was performed using the SMARTer RACE 5′/3′ Kit (Clontech Laboratories, USA). The reaction conditions were as follows: 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 68°C for 1 minute, 72°C for 1 minute, and final step of 72°C for 10 minutes. The amplicons were ligated into pRACE vectors and transformed into Stellar Competent Cells (Clontech Laboratories). Plasmid DNA of positive clones was then extracted and purified by Wizard Plus SV Minipreps DNA Purification System (Promega, USA). Three independent clones were sequenced by First BASE Laboratories Sdn Bhd (Malaysia) and showed the same result.