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.
Sybr qpcr supermix plus
Manufactured by Takara Bio
Sourced in Japan
SYBR qPCR Supermix Plus is a ready-to-use mixture for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including a thermostable DNA polymerase, SYBR Green I dye, and optimized buffer system, to perform qPCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cdna synthesis supermix, by Takara Bio (2 mentions)
Taqman universal pcr master mix, by Takara Bio (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr qpcr supermix plus, by Bio-Rad (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
3 protocols using sybr qpcr supermix plus
1
Quantifying Transcriptional Responses to Fungal Stress in Grapevine
2
Quantitative Real-Time PCR (qRT-PCR) Protocol
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qRT-PCR was conducted as described previously.18 (link) The whole brain or PAs were homogenized and mixed with Trizol reagent for RNA extraction. The yields of RNA were assessed using a Nanodrop 2000 spectrophotometer. cDNA synthesis was conducted using a cDNA Synthesis Supermix (TAKARA, Japan). qRT-PCR assays were performed using the SYBR qPCR Supermix Plus (TAKARA, Japan). β-actin was used to normalize gene expression data. To detect miRNA expression, total RNA was reverse transcribed and then mixed with TaqMan Universal PCR Master Mix (TAKARA, Japan) and miRNA-specific TaqMan primers (Springen, Nanjing, China). MiRNA expression data were normalized to U6 RNA. The fold-change of gene expression was measured using the 2−ΔΔCt method.18 (link) All primer sequences for qRT-PCR are presented in Supplementary Table S1
3
RNA Extraction and qRT-PCR Analysis
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Whole brains or astrocytes were homogenized and dissolved in Trizol (Invitrogen, Carlsbad, CA, USA) to extract RNA. The concentrations of RNA were measured by a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). Reverse transcription was performed using a cDNA Synthesis Supermix (TAKARA, Kusatsu, Shiga, Japan). The cDNA was subjected to qRT-PCR with the SYBR qPCR Supermix Plus (TAKARA). Expression data were normalized to β-actin. In addition, the total RNA was reverse transcribed to determine the miRNA expression, and the resulting cDNA was mixed with miRNA-specific TaqMan primers (springen, Nanjing, China) and TaqMan Universal PCR Master Mix (TAKARA). U6 RNA was used as an endogenous control for data normalization. Relative changes in expression were measured using the comparative threshold cycle (Ct) method and 2-ΔΔCt as described,21 (link) and the results indicated the fold change of expression. These primers for qRT-PCR are shown in Supplementary Table S1
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