The total RNA of the fruit peel and flesh samples were extracted using the Total RNA kit (Aidlab Biotechnology, Beijing, China). The quality and concentration of the RNA were detected by agarose gel electrophoresis and Nanodrop spectrophotometer. The cDNA was synthesized by TRUEscript RT MasterMix (OneStep gDNA Removal) kit (Aidlab Biotechnology, Beijing, China). The qRT-PCR was performed according to the method described in our previous report (Ye et al., 2022 (link)). The primers used in this study were listed in Table S8
Total rna kit
Manufactured by Aidlab
Sourced in China
The Total RNA Kit is a laboratory product designed for the extraction and purification of total RNA from various biological samples. It provides a reliable and efficient method for obtaining high-quality RNA that can be used in downstream applications such as qRT-PCR, RNA-seq, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green qpcr mix, by Aidlab (2 mentions)
Cdna synthesis kit, by Aidlab (2 mentions)
Abi 7500 real time system, by Thermo Fisher Scientific (2 mentions)
Truescript rt mastermix, by Aidlab (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
6 well plate, by Corning (1 mentions)
Pmd18 t, by Takara Bio (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Sybr green pcr kit, by Bio-Rad (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Six well plate, by Corning (1 mentions)
7 protocols using total rna kit
1
RNA Extraction and Quantification Protocol
2
Transcriptional Profiling of Kiwifruit Flowers
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The flowers samples were collected from the male and female plants of the same hybrid generation which were 6 years old and grow very well in the National Kiwifruit Germplasm Resource Garden of China, located in the Research Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences (30°29’ N, 114°16’ E), Wuhan, China. The male and female flowers were sampled at stage 4 when flowers begin to differentiate (Caporali et al., 2019 (link)), each sample containing three biological replicates. The sepals, petals, pistils, stamens, and ovaries of male and female flowers were separated and frozen in liquid nitrogen immediately, and then stored at −80°C. According to the manufacturer’s instructions, the total RNA was extracted by Total RNA kit (Aidlab Biotechnology, China) and the first strand cDNA was synthesized by SuperScript™ IV VILO™ Master Mix (Thermo Fisher, China). The qRT-PCR was performed in Applied Biosystems 7500 Real-Time PCR System with the reaction system as follows: 5 μL of Hieff qPCR SYBR Green Master Mix (YEASEN, Shanghai, China), 0.2 μL each for forward and reverse primers, 4.1 μL of ddH2O, and 0.5 μL cDNA. The 2−ΔΔCT method was used to calculate the relative gene expression of AcMADS-box genes (Arocho et al., 2006 (link)). The AcActin gene (Acc05529.1) was used for normalization of qRT-PCR data. The primers are listed in Supplementary Table S9 .
3
Extraction and cDNA Synthesis Protocol
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Total RNA was extracted from the young capitula at different developmental stages using Total RNA Kit (Aidlab Biotechnologies Ltd., Beijing, China). First-strand cDNA was synthesized using cDNA synthesis (Takara Bio Inc., Shiga, Japan). Genomic DNA was extracted from different developmental stages of the apical bud by the modified CTAB-based method [33 ].
4
Caco-2 Cells Regulation of SGLT1 and GLUT2
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The Caco-2 cells were seeded in 6-well plates (Corning Costar Co., NY, USA) at a density of 4 × 10 4 cells/cm 2 and were cultured for 21 days. After incubation with different tea catechins (200 μM in full culture medium) for 2 hours or 24 hours, the total RNA from the Caco-2 cells was isolated with a Total RNA Kit (Aidlab, Beijing, China). First-strand cDNA was synthesized using a cDNA synthesis Kit (Aidlab, Beijing, China). The gene expression of SGLT1 and GLUT2 was examined by quantitative real-time PCR with the SYBR Green qPCR Mix (Aidlab, Beijing, China) in an ABI 7500 Real-Time System (Applied Biosystems, USA). The reaction was carried out in a 10 μL RT-reaction mixture containing 5 μL of 2× SYBR Green qPCR Mix, 0.2 μL of forward and reverse primers (10 μmol/μL), and de-ionized water to a final volume of 10 μL, and was performed under the following cycling conditions: an initial denaturing step of 10 minutes at 95°C, followed by 40 cycles consisting of 5 seconds at 95°C and 34 seconds at 60°C.
All of the qPCR experiments were repeated three times. The data were analyzed using the 2 -ΔΔCT method. 35 All of the primers were synthesized by Sangon Biotech (Shanghai, China), and their sequences are depicted in Table 1.
All of the qPCR experiments were repeated three times. The data were analyzed using the 2 -ΔΔCT method. 35 All of the primers were synthesized by Sangon Biotech (Shanghai, China), and their sequences are depicted in Table 1.
5
Cloning and Sequencing of ALOG Genes
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To further confirm the identified genes, full-length genomic DNA sequence and complete CDS of P. hybrida line W115 ALOG genes were amplified using gene-specific primers. Mixture of different tissues was used to extract total RNA with RNA pure Total RNA Kit (Aidlab, China). 2 μg of RNA was used to synthesize the cDNA using cDNA synthesis Kit (Takara, Japan) with Oligo dT Primer and Random 6 mers. Then 2 μl of cDNA dilution (1:20) was added in PCR system with the 2 × High-Fidelity Master Mix DNA Polymerase (Tsingke, China). The products of PCR were purified with Axyprep DNA Gel Extraction Kit (Axygen, USA), and then cloned into pMD18-T (Takara, Japan) followed by heat shock into E. coli DH5α. For each gene, three positive clones were picked for sequencing (Augct, China).
6
Quantitative Analysis of lncRNAs and Anthocyanin Genes
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Total RNA was extracted from the CK, TRV-LNC1 and TRV-LNC2 fruits using the Total RNA Kit (Aidlab, Beijing, China). Briefly, the first cDNA strand was obtained using the Prime Script™ RT Master Mix (Takara, Dalian, China). All primers used in this study are listed in Supplementary Table S1 . qRT-PCR was performed on the Bio-Rad CFX96 Touch™ RealTime PCR Detection System using a standard SYBR Green PCR Kit (Bio-Rad). The conditions for quantitative PCR and the primers for the lncRNAs and genes are listed in Supplementary Table S1 . Sample cycle threshold (Ct) values were determined and standardized relative to the endogenous control gene 18S rRNA, and the 2–ΔΔCT method was used to calculate the relative changes in gene expression based on the qRT-PCR data. All reactions were carried out in three biological repetitions. qRT-PCR was used to validate the sequencing results of two miRNAs and the genes involved in anthocyanin biosynthesis. The quantitative PCR conditions and the primers for the miRNAs and genes are listed in Supplementary Table S1 .
7
Quantifying MRP2 Gene Expression in Caco-2 Cells
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The Caco-2 cells were seeded in six-well plates (Corning Costar Co., NY, USA) at a density of 4 × 104 cells/cm2, and were cultured for 21 days. After incubation with each tea catechins (300 μmol/L in full culture medium) for 2 h or 24 h, the total RNA from the Caco-2 cells were isolated with a Total RNA Kit (Aidlab, Beijing, China). First-strand cDNA was synthesized using a cDNA synthesis Kit (Aidlab, Beijing, China). The gene expression of MRP2 was examined by quantitative real-time PCR with the SYBR Green qPCR Mix (Aidlab, Beijing, China) in an ABI7500 Real-Time System (Applied Biosystems, USA). The reaction was carried out in a 10 μL RT-reaction mixture containing 5 μL 2 × SYBR Green qPCR Mix, 0.2 μL forward and reverse primers (10 μmol/μL), and water to a final volume of 10 μL, and was performed under the following cycling conditions: an initial denaturing step of 10 min at 95 °C, followed by 40 cycles consisting of 5 s at 95 °C and 34 s at 60 °C.
All of the qPCR experiments were repeated in three biologicals. The data were analyzed using the 2-ΔΔCT method [39 (link)]. All of the primers were synthesized by Sangon Biotech (Shanghai, China), as previously described [40 (link)], and their sequences are depicted inTable 3 .
All of the qPCR experiments were repeated in three biologicals. The data were analyzed using the 2-ΔΔCT method [39 (link)]. All of the primers were synthesized by Sangon Biotech (Shanghai, China), as previously described [40 (link)], and their sequences are depicted in
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