Sybr qpcr supermix plus

Complementary DNA (cDNA) libraries were synthesized from 1 μg total RNA by the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan). Subsequently, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was carried out with SYBR qPCR SuperMix Plus (Takara, Kyoto, Japan) on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) on 12 genes selected among the DEGs that respond to D. seriata 98.1 incubation, in a total volume of 10 μL containing 5 μL SYBR qPCR SuperMix Plus, 0.2 μL each primer at 0.2 μM and 1 μL cDNA synthesized above. The thermal cycling consisted of a hold at 95 °C for 1 min, followed by 45 cycles of 95 °C for 20 s, 58 °C for 20 s and 72 °C for 30 s, and then a melting curve program at 65 to 95 °C raised gradually by 0.5 °C every 5 s. The expression of grapevine actin was amplified as an internal control. The relative expression levels compared to the mock-incubated control were calculated using the normalized expression method (2^−ΔΔCT). The primers used in this experiment of each selected gene are listed in Supplementary Table S1. Standard errors of the mean values were generated by two biological replicates each comprising three technical replicates conducted for each gene.

To validate the transcriptome data and to characterize the flavonoid genes that were differentially expressed in the developing seeds of yellow- and dark-seeded B. juncea, total RNA was isolated from the samples using a DNA away RNA Mini-Prep Kit (Sangon Biotech, Shanghai, China). Subsequently, the cDNAs were synthesized using an RNA PCR Kit (AMV, v3.0) based on the manufacturer’s protocols (Takara, Dalian, China). The cDNA was subjected to RT-qPCR analysis using SYBR qPCR SuperMix Plus (NovoStart) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA), as previously described [13 (link)]. Tonoplastic intrinsic proteins-41 (TIPS-41) was used as a reference gene to normalize the gene expression levels via the 2^−∆∆Ct method [90 (link)]. Three biological replicates were performed for all experiments. The specific primer sequences used in this study were obtained from the RT-qPCR Primer Database [91 (link)] and are listed in Supplementary Table S10.