Sybr green realtime pcr master mix plus kit

Quantitative real-time PCR (qRT-PCR) analysis was performed to determine the transcript abundance of the GmWRKY31 gene using a SYBR^® Green Real Time PCR Master Mix Plus kit according to the manufacturer’s instructions (TOYOBO, Japan) on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, USA). The primers was GmWRKY31-qF/R (Supplementary Table S1). Total RNA was isolated from soybean leaves using TRIzol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Reverse transcription was performed using 1 μg of total RNA and a ReverTra Ace^® qPCR RT Kit (TOYOBO, Japan). The PCR protocol was 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The amplification product was confirmed by melting curve analysis in one-degree intervals from 95 to 60°C. The relative expression value was calculated by the 2^-ΔΔCT method using the soybean internal control gene GmEF1b (GenBank accession no. NM_001248778) with the primers GmEF1b-F/R (Supplementary Table S1). Each qRT-PCR was performed on three biological replicates with three technical replicates each.

The cells were harvested, and the total RNA was isolated using TRIzol^® reagent (Life Technology, Carlsbad, CA, USA) and quantified using a spectrophotometer (DeNovix DS-11, Wilmington, USA). Then, the RNA was reverse transcribed into cDNA using the ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan). PCR amplification was performed using a SYBR^® Green Realtime PCR Master Mix-Plus-Kit (TOYOBO, Osaka, Japan) on a 7500 Real Time PCR System (Applied Biosystem®, Life Technology). More information regarding the primers and probes is provided in Additional file 1.

Total cellular and viral RNA were isolated using phenol/chloroform extraction and reverse transcribed using a random primer with the ReverTra Ace Alpha kit (Toyobo, Dalian). Real-time PCR quantification was performed using the SYBR Green Real-time PCR Master Mix (Plus) kit (Toyobo, Dalian). Relative HCV genome RNA levels were calculated by the 2^−ΔΔC_T method with GAPDH mRNA as an internal control and were shown as relative change by normalizing to the untreated control samples (Primer pairs RT-HCV and RT-GAPDH respectively, Supplementary Table S1).

Total RNA was extracted from approximately 20,000 mixed-stage nematodes of Ab-S24 and Ab-N10 populations using the Invitrogen TRIzol® Reagent kit. RNA from different development stages of the Ab-S24 population was extracted from 500 each of females, males, juveniles and eggs using a MicroElute Total RNA kit (Omega, USA). The extracted RNA was reverse transcribed into cDNA using the RQ1 RNase-Free DNase (Promega) reverse transcription kit as described above. The expression levels of Ab-cb-1 in the Ab-S24 and Ab-N10 populations and in four different development stages of the Ab-S24 population were detected on a CFX-96 (Bio-Rad) qPCR machine with cDNA as a template using a SYBR Green Real-time PCR Master Mix Plus kit (Toyobo, Japan). Specific primers qPCRC-F and qPCRC-R (Table 1) were designed to detect Ab-cb-1 expression. A 140 bp sequence of 18S rRNA (AY508035) was amplified as a reference gene using the primers 18sF and 18sR (Table 1). The qPCR data were analyzed using the CFX Manager software provided by Bio-Rad. All experiments were performed in three biological replicates and each in three replicates.