The expression of 15 differentially expressed genes were validated using qRT-PCR. The extraction of total RNA from fruit flesh was carried out using a modified CTAB method (Xuan et al., 2015 ). All primers used for qRT-PCR were provided in Supplementary Table 1 . The synthesis of cDNA was carried out using the PrimeScript® RT reagent kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was performed using the Eppendorf Realplex4 real-time PCR system (Hamburg, Germany) as described previously (Fang et al., 2016b (link)). qRT-PCR conditions were 5 min at 95°C, followed by 40 cycles of 5 s at 95°C, 15 s at 60°C, and 30 s at 72°C, followed by 60–95°C melting curve detection. Actin (PsSY0026636) gene was used as the reference. The expression levels were calculated as described previously (Fang et al., 2016b (link)). Three biological and three technical replications were performed.
Realplex4 real time pcr system
Manufactured by Eppendorf
Sourced in Germany
The Realplex4 real-time PCR system is a laboratory instrument designed for quantitative polymerase chain reaction (qPCR) analysis. The device is capable of performing real-time detection and quantification of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Avian myeloblastosis virus reverse transcriptase, by Promega (1 mentions)
Sybr green supermix, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hiscript 3 rt supermix for qpcr with gdna wiper, by Vazyme (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
E z n a plant rna kit, by Omega Bio-Tek (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
4 protocols using realplex4 real time pcr system
1
Validating Differential Gene Expression
2
Quantifying BTBD10 and Cyclin D1 Expression
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Total RNA was extracted using Trizol reagent (Invitrogen, USA), and cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Promega, USA) in accordance with the manufacturer’s instructions. PCR was performed on a RealPlex4 Real-time PCR system (Eppendorf, Germany) using SYBR Green Supermix (Takara Bio Inc., Japan). The following primers were synthesized at Sangon Biotech (China): BTBD10, 5′-AGCAGTGCTGGGAACAGCAGCAG-3′ (forward) and 5′-TATATTCCGAGCTCCTT-3′ (reverse); 18 S rRNA, 5′-CAGCCACCCGAGATTGAGCA-3′ (forward) and 5′-TAGTAGCGACGGGCGGTGTG-3′ (reverse); cyclin D1, 5′-AGGAGAACAAACAGATCA-3′ (forward) and 5′-TAGGACAGGAAGTTGTTG-3′ (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-CCACCCATGGCAAATTCCATGGCA-3′ (forward) and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (reverse). Relative mRNA expression was calculated using the 2−ΔΔCT method. The expression of BTBD10 was normalized to that of 18 S rRNA, and the expression of cyclin D1 was normalized to that of GAPDH.
3
Quantification of Berry Transcripts by qRT-PCR
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Total RNA was extracted from green, red and black Wufanshu berries using EZNA Plant RNA Kit (Omega Bio-tek). First strand cDNA was prepared from 500 ng total RNA HiScript III RT SuperMix for qPCR with gDNA wiper (Vazyme, Nanjing, China). qRT-PCR was performed using the Eppendorf Realplex4 real-time PCR system (Hamburg, Germany) in a total volume of 20 μL in each well containing 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 6 μL of cDNA (in 1:30 dilution), and 0.4 μL 10 μM primers. qRT-PCR conditions were 30 s at 95°C, followed by 40 cycles of 5 s at 95°C, 15 s at 60°C, followed by 60°C to 95°C melting curve detection. Actin gene (c87909.graph_c0) was used as the reference. The expression levels were calculated using the 2–ΔΔCT method. Three biological and four technical replications were performed. Primers for qRT-PCR were listed in Supplementary Table 1 . Linear regression analysis of FPKM and qPCR was performed using Minitab® 18.
4
Anthocyanin Biosynthesis Gene Validation
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Nineteen candidate differentially expressed genes involved in anthocyanin biosynthesis were selected for validation by real-time quantitative RT-PCR (qRT-PCR). Total RNA from fruit samples was extracted using a modified CTAB method as described by Xuan et al. (2015 (link)). The primer sequences used for qRT-PCR are listed in Table S1 . The cDNA was transcribed from 500 ng of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China). Quantitative RT-PCR was performed using the Eppendorf RealPlex4 real-time PCR system (Hamburg, Germany) in a total volume of 20 μL in each well containing 10 μL of 2 × SYBR Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa), 1 μL of cDNA (in 1:10 dilution), and 0.4 μL 10 μM primers. Quantitative PCR conditions were 5 min at 95°C, followed by 40 cycles of 5 s at 95°C, 15 s at annealing temperatures listed in Table S1 , and 30 s at 72°C, followed by 60–95°C melting curve detection. The actin gene was used as the reference. The expression levels were calculated as described by Fang et al. (2013 (link)). Three biological and three technical replicates were performed.
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