Uniq 10 trizol rna extraction kit

Stains grew in 40 mL of SC-SCAA medium at 30 °C at 200 rpm from OD 0.2 to OD₆₀₀ 0.6, and cells were harvested and washed. Total RNA was extracted using the UNIQ-10 Trizol RNA extraction kit (Sangon Biological Engineering, China). The PrimeScript RT-PCR Kit (Takara, Japan) was used for cDNA synthesis. The relative transcription levels of the genes were quantified by real-time quantitative PCR using the SYBR Green Master Mix Kit (Roche Molecular Biochemicals, Germany). HAC1 mRNA splicing were detected through PCR using cDNA as the template.

Total RNA was prepared from 40 mL exponentially growing cell cultures using the UNIQ-10 Trizol RNA extraction kit (Sangon Biological Engineering, Shanghai, China). The samples were digested using DNase I treatment (TakaRa, Dalian, China) to avoid DNA contamination. The treated total RNA was used for cDNA synthesis using the PrimeScript RT-PCR Kit (TakaRa, Dalian, China). qPCR was performed using the SYBR Green Master Mix Kit (Roche Molecular Biochemicals, Germany). The ACT1 gene was chosen as the internal control gene. The relative transcription levels of genes were analyzed using the 2^−ΔΔCT method.

The mth-like genes were searched using the D. melanogaster mth sequence as a query in the sequences from the adult D. helophoroides transcriptome database (previously published under the accession GBCX00000000) that had already been identified and annotated by Blastx [28 ] against the non-redundant (NR) protein database at the National Center for Biotechnology Information (NCBI) with a cut-off E-value of 1.0 × 10⁻⁵ [27 (link),29 ]. Based on the cDNA fragments obtained by searching the transcriptome annotation files, gene-specific primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) to amplify the full length mth-likes of D. helophoroides. The primers used for PCR-based cloning are shown in Table 1. The rapid amplification of cDNA ends PCR (RACE-PCR) technique was applied to obtain the full-length mth-like cDNAs with a SMARTer™ RACE cDNA Amplification Kit (Clontech, Beijing, China) according to the manufacturer’s instructions. Total RNA isolated from adult insects using a UNIQ-10 Trizol RNA extraction kit (Sangon, Shanghai, China) was used to obtain cDNA templates needed in RACE-PCR. The PCR products were cloned into the pMD-19-T Vector (TaKaRa, Kyoto, Japan) and sequenced by Sangon Biotech (Shanghai, China).