Revertra ace reverse transcriptase

A real-time quantification assay for miRNA was conducted as previously described[21 (link)]. Briefly, the assay was performed using stem-loop RT followed by quantitative PCR. First, 1 μg total RNA was reverse-transcribed to cDNA using ReverTra Ace reverse transcriptase (Toyobo Co.,Osaka, Japan) and miRNA-specific stem-loop RT primer. The mix was incubated at 37°C for 15 min, 85°C for 5 min and then held at 4°C using an Applied Biosystems 9700 Thermocycler. Then, quantitative PCR was performed on the Agilent TechnologiesMx3000P /Mx3005P Real-Time PCR Detection System by using a standard SYBR Green Real-time PCR Master Mix(Toyobo: QPK-201). In each reaction, 25 μL reaction mixtures containing 1 μL cDNA (1: 10 dilution) were prepared and incubated at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s in a 96-well optical plate. The melting curve analysis and agarose gel electrophoresis were used to confirm the specific PCR products. All reactions were run in triplicate and porcine U6 snRNA was used as an endogenous reference. To calculate the expression level differences of miRNAs between samples examined, the △△Ct method was used [22 (link)]. Three independent samples were analyzed for each rat.

RNA samples of 12 piglets used for the RNA-seq experiment were analyzed by quantitative real-time PCR (RT-qPCR). cDNA synthesis was performed using ReverTra Ace reverse transcriptase (Toyobo Co., Osaka, Japan) following the manufacturer’s instruction. RT-qPCR was performed using a StepOne Plus real-time PCR system with SYBR Green master mix (Applied Biosystems, Singapore). Specific primers were designed according to the sequences of selected transcripts using the NCBI (Supplemental Table S1). Each real-time RT-PCR reaction in 20 μL of reaction mixture comprised 10 μL of KOD SYBR^® qPCR Mix (TaKaRa, Dalian, China), 0.4 μL of 50 × ROX reference dye, 0.2 μL of each primer, 2 μL of cDNA, and 7.2 μL of RNase-free dH₂O. Swine 18S rRNA was used as an internal control gene, and all samples were analyzed in triplicate. The relative expression levels of genes were determined using 2^−ΔΔCt value methods. All data were analyzed by Student’s t-test using SPSS version 21.0 statistical software. RT-qPCR data were presented as the mean ± standard deviation, and p < 0.05 was considered statistically significant.

Total RNA was extracted by using RNAiso Plus (Takara-Bio, Kyoto, Japan) according to the manufacturer’s instructions. The quality and concentration of total RNA were determined spectrophotometrically by using a NanoDrop Lite UV-Vis Spectrophotometer (Thermo Fisher Scientific). Total RNA (1 μg) was reverse-transcribed to cDNAs by using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan) and random hexamer primers (Takara-Bio) according to the manufacturer’s protocols. Quantitative PCR was performed in an Applied Biosystems 7500 Real Time PCR System (Thermo Fisher Scientific) using Power SYBR Green Master Mix (Thermo Fisher Scientific) and the primer sets (Table 1). The comparative Ct method was employed to calculate the relative mRNA level of the desired gene [40 (link)], and values were normalized to that of the TATA-binding protein (TBP) gene.

Thalli or antheridiophores frozen in liquid nitrogen were ground with a mortar and a pestle into fine powder. Total RNA was extracted from the tissue powder using the RNeasy Plant Mini Kit (Qiagen). cDNA was synthesized using ReverTra Ace reverse transcriptase (Toyobo). Amplification was performed with GoTaq DNA polymerase (Promega).

Total RNA was isolated from rice tissue using Trizol (Invitrogen). For quantitative RT-PCR, total RNA was treated with DNA remover to remove contaminating genomic DNA. cDNA was synthesized using ReverTra Ace reverse transcriptase (Toyobo, Japan). To determine Pb1 expression, quantitative RT-PCRs were run on a Thermal Cycler Dice TP800 system (Takara Bio, Japan) as shown previously (Hayashi et al. 2010 (link)) using primers Pb1sp4Fw/Pb1s4Rv. Rice ubiquitin 1 (Rubq1; AK121590) was used as an internal standard. The primers used in quantitative RT-PCR were listed in Additional file 1: Table S1.