Cf96 real time pcr detection system

Total RNA was extracted from fruit at each stage of development (1–8 weeks). The first‐strand cDNA products were used as templates for PCR with specific primers (Table S3). Actin was used as an internal control. The PCR products were separated on 1% (w/v) agarose gels, stained with EtBr and visualized with a gel imaging system. To investigate the transcriptional patterns of various structural genes in the anthocyanin synthesis pathway at different stages of fruit development, qRT‐PCR analyses were performed with SYBR^® Premix Ex Taq II (Bio‐Rad, Hercules, CA) with a Bio‐Rad CF96 Real‐Time PCR Detection System. The specific primers for the structural genes are shown in Table S3. The reaction mixture (10 μL total volume) contained 5 μL SYBR Premix (2×), 1.0 μL forward primer (10 μm), 1.0 μL reverse primer (10 μm), 1 μL cDNA template (20 ng) and 2.0 μL ddH₂O. The cycling conditions were as follows: denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 55 s and 60°C for 30 s. Each reaction was performed in biological triplicate. Data were analysed using the 2−ΔΔCT method (Livak and Schmittgen, 2001). The transcript levels of specific genes were normalized to that of Actin.

Quantitative RT-PCR (Q-PCR) was used to measure mRNA and microRNA (miRNA) expression levels (primers shown in Supplementary Table S1). Total RNA was extracted from adipose tissues using TRIzol reagent (Invitrogen) and further purified with RNeasy columns (Qiagen) according to the manufacturer’s protocol. Reverse transcription of mRNA and miRNA was performed using the PrimeScript RT Master Mix kit and the PrimeScript^™ miRNA RT-PCR Kit, respectively (both obtained from TaKaRa, Dalian, China) following the manufacturer’s recommendations. Quantitative PCR was performed using the SYBR Green Real-time PCR Master Mix (TaKaRa) on a CF96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). The ACTB, TBP and TOP2B genes were simultaneously used as internal controls for mRNA normalization. Expression levels of U6 served as endogenous controls for miRNA expression and were utilized to normalize the corresponding data. The 2^−ΔΔCt method was used to determine the relative mRNA and miRNA abundance30 (link).

Quantitative RT-PCR (q-PCR) was used to measure mRNA and microRNA (miRNA) expression. Total RNA was extracted from adipose tissue and cell samples using Trizol Reagent (Invitrogen, Carlsbad, CA, United States), and was further purified with RNeasy columns (Qiagen, CA, United States) according to the manufacturer’s protocol. Subsequently, mRNA and miRNA were reverse-transcribed to cDNA using the PrimeScript^TM RT Master Mix Kit and the PrimeScript miRNA RT-PCR Kit, respectively (TaKaRa, Dalian, China). Quantitative PCR was performed using the SYBR Green Real-time PCR Master Mix (TaKaRa, Dalian, China) by CF96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, United States). β-actin and U6 were served as endogenous controls for mRNA and miRNA expression, respectively. The 2^−ΔΔCt method was used to calculate the relative expression levels of mRNA and miRNA (Livak and Schmittgen, 2001 (link)). All PCR primer sequences were shown in Table 1.

The quantitative RT-PCR (Q-PCR) was performed according to our previously described study [59 (link)]. Briefly, SYBR Green Real-time PCR Master Mix (TaKaRa, Dalian, China) was used to perform the reactions on CF96 Real-Time PCR Detection System (Bio-Rad). The 2^−ΔΔCt method was used to determine the relative mRNA abundance. ACTB, TBP, and TOP2B genes were simultaneously used as internal gene for normalization. The ratio of mitochondrial genes (ATP6, COX2, and ND1) to nuclear DNA single copy gene (GCG) within the same samples was used to calculate the relative mtDNA copy number. All reactions were performed in triplicate. All PCR primer sequences are shown in Supplementary Table S9.

The relative mtDNA copy number was determined by quantitative polymerase chain reaction (qPCR). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the SYBR Green Real-time PCR Master Mix (Takara, Dalian, China) on a CF96 Real-Time PCR Detection System (Bio-Rad, Richmond, CA, United States). The ratio of mitochondrial genes (ATP6 and COX2) to nuclear DNA single copy gene (GCG) within the same sample was used to calculate the mtDNA content (primer sequences are listed in Supplementary Table S3). All reactions were performed in triplicate. Relative mtDNA copy number per diploid cell was calculated by the 2^ΔCt method.