Sybr green qpcr mix

Total RNA from crabapple leaves, tobacco petals and apple peel were extracted as described above. DNase I (TaKaRa, Ohtsu, Japan) was added to remove genomic DNA, and the samples were then subjected to cDNA synthesis using the Access RT-PCR System (Promega, USA), according to the manufacturer’s instructions. The expression levels of flavonoid biosynthetic genes in crabapple and tobacco were analyzed using qRT-PCR and the SYBR Green qPCR Mix (TaKaRa, Ohtsu, Japan) and the Bio-Rad CFX96 Real-Time PCR System (BIO-RAD, USA), according to the manufacturers’ instructions. The PCR primers were designed using NCBI Primer BLAST and are listed in Table S1.
qRT-PCR analysis was carried out in a total volume of 20 μl containing 9 μl of 2 × SYBR Green qPCR Mix (TaKaRa, Ohtsu, Japan), 0.1 μM specific primers (each), and 100 ng of template cDNA. The reaction mixtures were heated to 95 °C for 30 s, followed by 39 cycles at 95 °C for 10 s, 50–59 °C for 15 s, and 72 °C for 30 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. The transcript levels were normalized using the Malus 18 S ribosomal RNA gene (DQ341382, for apple and crabapple) or the NtActin gene (GQ339768, for tobacco) as the internal controls and calculated using the 2^{(−∆∆Ct)} analysis method60 (link).

Total RNA from flower tissues was extracted using an EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China) according to the manufacturer's instructions. DNase I (TaKaRa, Japan) was added to remove genomic DNA, and the samples were subjected to cDNA synthesis using the Access RT-PCR System (Promega, USA) according to the manufacturer's instructions. RNA samples (1 μg) were reverse transcribed into complementary DNA (cDNA) using an oligo (dT) 18 primer and M-MLV reverse transcriptase (TaKaRa) following the manufacturer's protocol. qRT-PCR was carried out using SYBR® Premix Ex Taq™ II(Perfect Real Time) (TaKaRa) on the CFX96™ Real Time System (Bio-Rad). The differences in gene expression were calculated using the 2^{∧(−ΔΔCt)} analysis method, and the transcription levels were determined by relative quantification using the Malus 18S ribosomal RNA gene as the reference gene.
The qPCR analysis was conducted in a total volume of 20 μl containing 9 μl of 2 × SYBR Green qPCR Mix (Takara, Japan), 0.1 μM specific primers (each), and 100 ng of template cDNA. The reaction mixtures were heated to 95°C for 30 s, followed by 39 cycles at 95°C for 10 s, 59°C for 15 s, and 72°C for 30 s. The sequence and the information of the primers used in the qPCR are presented in Table S1 and Figure S2.

Total RNA extraction, cDNA synthesis and qRT-PCRs were performed according to Hu et al. (2016) [33 (link)] described. Briefly, total RNA was extracted from fresh tissues using Trizol reagent (Invitrogen, Carlsbad, CA). The first strand cDNA fragments were synthesized from 2 μg of total RNA using oligo(dT)12-18 primer using cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada) after RNA quality and integrity was checked by Nanodrop 2000 and 0.8% agarose gel. Gene-specific primers (Table 2) were designed based on the target gene sequences using Primer 5 software. The qRT-PCRs were performed with ABI7500 in a final volume of 20 μL, with each containing 2 μl of cDNA, 10 μL of 2×SYBR Green qPCR Mix (Takara, Otsu, Shiga, Japan) and 2 μM of the forward and reverse primers. Three independent biological replicates of each sample and two technical replicates of each biological replicate were used for real-time PCR analysis. The thermal cycling conditions were as follows: 40 cycles of 95°C denaturation for 5 s, and 52~55°C annealing and extension for 20 s. To determine relative fold differences for each sample, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the DDCT method as described by Livak and Schmittgen (2001) [40 (link)].

The miRNA expression levels were assessed by RT-qPCR. Total RNA was reverse transcribed to cDNA using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Beijing, China). RT-qPCR was carried out using a 2 × SYBR green qPCR mix (Takara, Beijing, China) and an ABI 7900HT sequence system (Thermo Fisher Scientific, Inc.). The reactions were incubated at 94°C for 3 min, followed by 40 cycles at 95°C for 15s and 62°C for 40s. The primer sequences for the four miRNAs are shown in Supplementary Table 1. U6 was used as an internal control for miRNAs. Statistical analyses of the results were performed using the 2^−∆∆CT relative quantification method.

Total RNA was extracted from Caco2 and SW480 cell lines using TRIzol reagent (Sangon Biotech, Shanghai, China), followed by reverse transcription into cDNA using a Prime Script RT Master Mix (Takara, Japan). Subsequently, qPCR was conducted using a 2× SYBR Green qPCR Mix (Takara, Japan) to detect the levels of the targeted mRNA, with GAPDH serving as the reference gene. The primer sequences used were as follows: ANKRD1 Forward primer: 5′-ATGTGGCGGTGAGGACTGG-3′, ANKRD1 Reverse primer: 5′-GTCGGATCATCTTATAGCGGTTCAG-3′, GAPDH Forward primer: 5′-ACCCACTCCTCCACCTTTGAC-3′, GAPDH Reverse primer: 5′-TCCACCACCCTGTTGCTGTAG-3′.

The expression of selected candidate genes was validated by quantitative real time PCR (qRT-PCR) using the same RNA samples as in the RNA-seq library construction. The first strand cDNA fragments were synthesized from 2 μg of total RNA using oligo(dT)_12–18 primer using cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada) according to the user manual. Gene-specific primers (Table 1) were designed based on the target gene sequences using Primer 5 software. UBI gene was used as internal standard. The qRT-PCRs were performed with ABI7500 in a final volume of 20 μL, with each containing 2 μL of cDNA, 10 μL of 2 × SYBR Green qPCR Mix (Takara, Otsu, Shiga, Japan) and 2 μM of the forward and reverse primers. Three independent biological replicates of each sample and two technical replicates of each biological replicate were used for real-time PCR analysis. The thermal cycling conditions were as follows: 40 cycles of 95 °C denaturation for 5 s, and 52 ~ 55 °C annealing and extension for 20 s. After the PCR, a melting curve was generated by gradually increasing the temperature to 95 °C to test the amplicon specificity. To determine relative fold differences for each sample, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the DDCT method as described by Livak and Schmittgen [59 (link)].