Taq universal sybr green supermix

Total RNA was isolated from OMAT and SCAT using TRIzol (Life Technologies, Grand Island, NY) and RT was performed as previously described [22 (link)]. Quantitative Real-time-PCR was performed using Taq universal SYBR Green Supermix (Bio-Rad). The primers used for ADM (Cat #: Hs00181605), CRLR (Cat #: Hs00173787), RAMP2 (Cat #: Hs00359352), and RAMP3 (Cat #: Hs00389130) are commercially available from Life Technologies, (Grand Island, NY). PCR primers used for amplification of lipid metabolism and proinflammatory molecules were purchased from Integrated DNA Technologies (IDT) and the primer sequences were list in Table 2. Amplification of GAPDH served as an endogenous control. PCR conditions for SYBR Green gene expression were 10 min at 95°C for 1 cycle, then 15 sec at 94°C, 30 sec at 60°C and 15 sec at 72°C for 39 cycles. All experiments were performed in triplicate. The average CT value was used to calculate the results using the 2–ΔΔCT method, and expressed in fold increase/decrease of the gene of interest.

Cells were lysed with a cell lysis buffer (10 mM Tris pH 7.4, 0.25% IGEPAL CA-630, and 150 mM NaCl). Cell lysates containing RNA samples were then instantly subjected to reverse transcription using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Taq Universal SYBR Green Supermix (Bio-Rad, USA) was used for quantitative PCR analysis. All primers used in this study were synthesized by BGI Genomics (Hong Kong) and the sequences are shown in Supplementary Table 3.

Quantitative Real-time PCR was performed using Taq universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) [23 (link)]. PCR primers used for amplification of ADM (Mm.PT.58.11111908), CRLR (Mm.PT.58.10636953), RAMP2 (Mm.PT.58.30553776), and RAMP3 (Mm.PT.58.8586280) were purchased from Integrated DNA Technologies (IDT, Coralville, IW, USA), and the primer sequences for insulin and ER stress genes were list in Table 1. Amplification of both β-actin and GAPDH served as endogenous housekeeping controls. PCR conditions for SYBR Green gene expression were 10 min at 95°C for 1 cycle, then 15 sec at 94°C, 30 sec at 60°C and 15 sec at 72°C for 39 cycles. All experiments were performed in triplicate, and the average CT value of the two housekeeping genes was used to calculate the results using the 2–ΔΔCT method and expressed in fold increase/decrease of the gene of interest.

The expression levels of a sRNA target gene were analysed by RT-qPCR. One to five ug of total RNAs from mock and infected B. napus leaf samples were converted to cDNA using the MMLV reverse transcriptase kit (Sigma-Aldrich). The cDNA samples were then diluted 1/20 before qPCR. The qPCR analysis was performed using the Bio-Rad Taq Universal SYBR Green Supermix according to the manufacturer’s instructions. The thermocycler settings were 95 °C for 2 min, then 95 °C for 15 s, 60 °C for 30 s and 72 °C for 15 s, and cycled for 40 times, followed by 72 °C for 2 min. Three biological and three technical replicates were used for each sample. Relative expression was calculated as per log (2^-∆Ct) method normalized to the B. napus housekeeping actin gene. The primers and adapters used for 5′ RACE and qPCR experiments are listed in Supplementary Table 9.

Quantitative Real-time-PCR was performed using Taq universal SYBR Green Supermix (Bio-Rad). PCR primers used for amplification for mitochondrial DNA (mtDNA)-encoded subunits of the electron transport chain were purchased from Integrated DNA Technologies (IDT) and the primer sequences were listed in Table 1. Amplification of 18S and GAPDH served as endogenous controls. PCR conditions for SYBR Green gene expression were 10 min at 95°C for 1 cycle, then 15 sec at 94°C, 30 sec at 60°C and 15 sec at 72°C for 39 cycles. All experiments were performed in triplicate. The average CT value was used to calculate the results using the 2–ΔΔCT method and expressed in fold increase/decrease of the gene of interest.

Overnight V. vulnificus cultures were adjusted to obtain an initial 2 × 10⁶ CFU/mL bacterial count. The WT strain was supplemented with or without secondary screening hit compounds or 2′,4′-DHC or DMSO in 3 mL fresh LBS medium at concentrations that were assessed as being non-growth inhibitory. The cultures were allowed to grow up to OD₆₀₀ ~1.8–2.0. The ∆hlyU V. vulnificus strain was included as the control. RNA was isolated using a QIAGEN RNeasy Mini kit and cDNA was synthesized using the Ecodry Premix (random hexamer) kit (Takara Bio, Mountain View, CA, USA) as described earlier [12 (link)]. The qRT-PCR reactions were performed using Taq Universal SYBR Green supermix (Bio-Rad, Hercules, CA, USA) to determine the relative expression of rtxA1, vvhA, hlyU, and hns genes wherein the gyrB gene served as an endogenous control. Relative gene expression was analyzed using the 2^−ΔΔCT method [32 (link)].