Primerscript first strand cdna synthesis kit

To validate RNA-seq results, the expression patterns of 21 randomly selected genes from various functional categories and regulation patterns were analysed by qRT-PCR at five different time points post infection (Additional file 1: Table S1). Three biological replicates were collected independently and immediately frozen in liquid nitrogen. Total RNA isolation was performed as described above, and 1 μg of total RNA was reverse-transcribed using PrimerScript First-Strand cDNA Synthesis Kit (Takara, Dalian, China). qRT-PCR was performed in a 25 μl reaction mixture containing 12.5 μl 2 × SYBR Master Premix ExTaq (Takara), 1 μl cDNA template (1:5 dilution), and 1 ul of each forward and reverse primer for the selected gene of interest. All primers were designed based on cDNAs using Primer Premier 5.0 (Additional file 1: Table S1). Three biological and three technical relipcates per sample were analysed using Bio-Rad Real-Time System (BioRad, Hercules, CA, USA) based on the 2^-ΔΔCT method [32 (link)]. qRT-PCR conditions were 95 °C for 2 min followed 40 cycles at 95 °C for 5 s, 60 °C for 30 s and 65 °C for 5 s. Actin11 (GeneBank: 209698678) was used as a reference gene.

Reverse transcription-PCR (RT-PCR) was performed by using PrimerScript First strand cDNA Synthesis Kit (TaKaRa, China). Viral RNA was reverse transcribed for 10 min at 30°C, 50 min at 42°C, and 5 min at 95°C. A single real-time multiplex PCR assay was followed for simultaneous detection of five enteric viruses: norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus (Jiang et al., 2014 (link)). The specific primers and probes used in this reaction were presented in Table 1. Each primer had a common tag sequence at the 5′ end of the sequence. A combination of two probes for Norovirus GI was designed in order to differentiate the five organisms under four detecting channels. The amplification reaction was consistent with previously described and cycling condition was the same as that of simultaneous detecting virulence loci of DEC or five enteric bacteria (Jiang et al., 2014 (link)).

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed (RT) using a the PrimerScript First Strand cDNA Synthesis Kit (TaKaRa) with random primers and oligo(dT) at the same time. BLAST searches against unigenes of cluster 3 and cluster 6 in A. euchroma transcriptome database resulted in two candidate genes, unigene18140 (AeHGO) and unigene32185, with sequence similarity to alcohol dehydrogenases (ADHs). Their full-length clones were acquired by PCR using the gene-specific primer pairs AeHGO-F1 (5′-TATGGAGCTCGGTACCATGGGAAATTCAGCAGAAC-3′) and AeHGO-R1 (5′- CGACAAGCTTGAATTCTTAGGCAGTTTTAAGAGTATTGC-3′), 32185-F1 (5′- TATGGAGCTCGGTACCATGTCCAACACTGCTGG-3′) and 32185-R1 (5′- CGACAAGCTTGAATTCTTAACCTTCCATGTTGATAATGC-3′). In these primers the extensions homologous to vector ends for subcloning were underlined. The PCR products were cloned into pEASY-Blunt Simple vector (TransGen) for sequencing and then subcloned into pCold II vector (Takara) using In-Fusion HD Cloning Kit (Takara), which resulted in the expression construct pCold II-AeHGO and pCold II-unigene32185.

The first-strand cDNAs were synthesized using a PrimerScript First Strand cDNA synthesis kit (TaKaRa, Japan) following the manufacturer’s protocol, in a total of 20 μl reaction volume including 1 μg of total RNA, 4 μl 5X PrimeScript RT Master Mix, and RNAase-free ddH₂O. The Semi-quantitative RT-PCR was performed in a final volume of 20 μl containing 2 μl of diluted cDNA, 10 μl 2X Premix Taq version 2.0 Mix (TaKaRa, Japan), and 200 nM of forward and reverse primers (Additional file 11: Table S8). The thermal cycling conditions were set as follows: different cycles of 95 °C for 30 s, 54.5–55 °C for 30 s (54.5, 55 °C for GmMATE75 and GmEF-1a, respectively), and 72 °C for 45 s. The number of cycles is based on the genes and designed primers, which is labeled in the results. The housekeeping gene GmEF-1α was used as the internal control [85 (link)]. Electrophoresis was performed using 1 % agarose gels.

All RNA from liver tissues was isolated using the Promega SV Total Isolation System (Promega, Madison, WI, United States) and quantified using a microplate reader. cDNA was synthesized using the PrimerScript first strand cDNA Synthesis Kit (Takara, Shiga, Japan). The mRNA expressions of key enzymes (CYP27A1, CYP7A1, and CYP8b1), transport (NTCP, bile salt excretion pump (BSEP), MRP2, MRP3, and MRP4), and metabolic enzymes (CYP) were detected using the Agilent Mx3005 P Real-Time PCR System (Agilent, Santa Clara, CA, United States) with the SYBR Green PCR kit. The thermal conditions were as follows: 95°C for 60 s, followed by 45 cycles at 95°C for 15 s and 60°C for 60 s. β-Actin was used as an internal control. All primers used for qRT-PCR were synthesized by Sangon Biotech (Shanghai, China) and are presented in appendix, section 3 (Supplementary Appendix S3).

Total RNA (50mg) was isolated with TRIzol Reagent (Invitrogen, 15596–026) according to the manufacturer’s protocol. RNA was dissolved in DEPC-treated water, and the integrity of the RNA was assessed by electrophoresis with a 1.0% agarose gel, and then the concentration and purity were examined at 260/230 and 260/280 absorbance ratios. Purified RNA was diluted to 1 mg/ml to synthesize first-strand cDNA using a Primer Script^™ First Strand cDNA Synthesis kit (TAKARA Bio Inc. Japan). The cDNA was used as the template for amplifying gene sequences and analyzing their expression. All primers used in this study were designed with Primer Premier 5.0 and are shown in S1 Table.