Mlv reverse transcriptase

The reverse transcription product with an oligo (dT) 24 primer and MLV reverse transcriptase (TaKaRa, Dalian, China) were used in PCR to amplify the target gene Oslaf-1 open reading frame according to the Takara (SYBR Premix Ex Taq II) manufacturer’s instructions. Based on the sequence of Oslaf-1 found in our laboratory, the cDNA of Oslaf-1 gene was obtained using a cDNA amplification kit (TaKaRa, Dalian, China). A pair of specific primers (Table 1) was designed to amplify the full-length cDNA using Primer Premier 5.0 software. The following reaction procedure was used for our PCR experiment for denaturing: 3 min at 95°C, 35 cycles for 30 s at 95°C, 30 s at 55°C, 30 s at 72°C, and 10 min at 72°C. The amplified product was cloned into a pMD18-T vector and transformed into the E. coli strain DH5α before sequencing.

Total RNA was isolated from the same samples used for the RNA-seq analysis using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. First-stand cDNA was synthesized using the Takara MLV-Reverse transcriptase (Takara Bio, Inc., Otsu, Japan) used for qRT-PCR analysis on the Bio-Rad CFX96 real-time PCR detection system with four biological replicates and using the SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan). Sixteen unigenes were randomly selected for the qRT-PCR analysis using gene-specific qRT-PCR primers (Additional file 3: Table S3). The 2^-ΔΔCT method [49 (link)] was used to calculate the relative expression level of each gene to the endogenous control Actin.

Total RNA was isolated from exponentially growing WT(pXMJ19), ΔsigH(pXMJ19) and ΔsigH(pXMJ19-sigH) strains exposed to different toxic agents of indicated concentrations for 30 min using the RNeasy Mini Kit (Qiagen, Hilden, Germany) along with the DNase I Kit (Sigma-Aldrich, Taufkirchen, Germany). Purified RNA was reverse-transcribed with random 9-mer primers and MLV reverse transcriptase (TaKaRa, Dalian, China). Quantitative RT-PCR analysis was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) as described previously [26] (link). The primers used are listed in S1 Table. The relative abundance of the target mRNAs was quantified based on the cycle threshold value. To standardize the results, the relative abundance of 16 S rRNA was used as an internal standard.

100 ng of total RNA was used for RT reacted in 25-μL reaction cocktails with and 50 μM of specific RT primers (Supplementary Table 2) that were denatured at 70 °C for 10 min, and then placed on ice. The RT buffer containing 200 U M-MLV reverse transcriptase (Takara), 40 U RNasin Ribonuclease inhibitor (Takara), and a low (10 μM) or high (1 mM) concentration of dNTPs were employed for an initial annealing step at 42 °C for 1 h and heated at 70 °C for 15 min. Then, the PCR reaction was terminated.

Total RNA was isolated from exponentially growing WT, ΔmsrR and ΔmsrR⁺ strains exposed to different toxic agents of indicated concentrations for 30 min using the RNeasy Mini Kit (Qiagen, Hilden, Germany) along with the DNase I Kit (Sigma-Aldrich, Taufkirchen, Germany). Purified RNA was reverse-transcribed with random 9-mer primers and MLV reverse transcriptase (TaKaRa, Dalian, China). Quantitative RT-PCR analysis (7500 Fast Real-Time PCR; Applied Biosystems, Foster City, CA) was performed as described previously [40 (link)]. The primers used were listed in Additional file 1: Table S2. To obtain standardization of results, the relative abundance of 16S rRNA was used as the internal standard.

Total RNA was isolated from exponentially growing strains exposed to different toxic agents of indicated concentrations for 30 min using the RNeasy Mini Kit (Qiagen, Hilden, Germany) along with the DNase I Kit (Sigma-Aldrich, Taufkirchen, Germany). Purified RNA was reverse-transcribed with random 9-mer primers and MLV reverse transcriptase (TaKaRa, Dalian, China). Quantitative RT-PCR analysis (7500 Fast Real-Time PCR; Applied Biosystems, Foster City, CA) was performed as described previously [20 (link)]. The primers used were listed in Additional file 1: Table S2. To obtain standardization of results, the relative abundance of 16S rRNA was used as the internal standard. The experiment was performed with at least three independent biological replicates.

Total RNA from A. sinica cysts (0 h) was extracted according to the kit instructions of RNAiso Plus (Takara, Dalian, China) and reverse transcribed into cDNA using an oligo (dT) primer and MLV reverse transcriptase (Takara). Specific primer sequences were designed based on the partial sequence of Artemia franciscana G1LEA and G3LEA and synthesized by Shenggong (Shanghai, China) using Premier 5.0 (Table 1). The PCR reaction conditions were as follows: initial incubation at 94C for 5 min; followed by 30 cycles of denaturation at 94C for 30 s, annealing at 55C for 30 s, elongation at 72C for 1 min; and a final incubation at 72C for 10 min. The full-length cDNAs of As-g1lea and As-g3lea were obtained using 3′-RACE (Takara) and 5′-RACE kits (Clontech, Chicago, USA), according to the manufacturer's instructions. The resultant PCR products were recovered and cloned into the PEASY-T1 vector (Quanshijin, Beijing, China) and sequenced. The 3′ and 5′ fragments were spliced together to obtain the full-length cDNA sequence using DNAman 6.0 (Lynnon Biosoft). The nucleotide sequence was submitted to GenBank (As-g1lea GenBank accession number: AMQ80946.1, As-g3lea GenBank accession number: KU851934).