Taqman reverse transcription regents

Intact RNA was converted to cDNA by reverse transcription using TaqMan Reverse Transcription Regents (Applied Biosystems, Foster City, CA, USA), according to protocols from the manufacturer. The final concentration of cDNA was 10 ng/ml verified by NanoDrop Spectrophotometer ND-1000. The samples were stored at −20°C until further used.

Total RNA was extracted from the juice sacs according to the method described by Ma et al. (2022) [43 (link)]. The RNeasy Mini Kit (Qiagen, Germany) was used to clean the extracted total RNA using on-column DNase digestion. The cDNA was synthesized with 2 µg of purified total RNA using a TaqMan Reverse Transcription Regents (Applied Biosystems, USA).
The gene expression was conducted by real-time quantitative PCR according to the method of Ma et al. (2022) [42 (link)]. As an endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used. The real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) on a StepOnePlus™ system (Applied Biosystems). Each reaction contained template cDNA, 900 nM primers, and a 250 nM probe (Table S2). The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The data obtained from the StepOnePlus™ real-time PCR software (Applied Biosystems) was used to analyze the gene expression. The results were normalized with the results of 18 S ribosomal RNA. The Real-time quantitative RT-PCR was performed in three replicates for each sample.

Total RNA was extracted from the flavedo of Satsuma mandarin and Ponkan mandarin according to the method described by Ikoma et al.^{47 (link)}. The total RNA was purified using a RNeasy Mini Kit (Qiagen, Germany) and treated with DNase (Takara, Japan) digestion on the column. The cDNA was synthesized from 600 ng of purified RNA and a random hexamer primer at 37 °C for 60 min using TaqMan Reverse Transcription Regents (Applied Biosystems, USA).
Real-time PCR was performed to investigate the expression of CitCHS1, CitCHS2, CitCHI, CitFNS, CitF3′H, CitF6H, and CitOMT. TaqMan probes and sets of primers were designed based on the common sequences with Primer Express software (Supplementary Table S2, Applied Biosystems, USA). The reaction of real-time PCR was performed with cDNA template, 900 nM primers, and 250 nM TaqMan MGB probe. The thermal cycling conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The levels of gene expression were analyzed using ABI PRISM 7300 Sequence Detection System Software (Applied Biosystems, USA) and normalized with the result of 18S ribosomal RNA. Real-time PCR was performed in three replicates for each sample, and the mean values and the standard error were calculated.