Abi prism 7300 sequence detection system software

Total RNA was isolated from treated cells by using Trizol Reagent (Invitrogen, CA, USA) and was treated with RNase-free DNase. Reverse transcription was performed with the cDNA reverse transcription kit (Fermentas, MA, USA). For real-time PCR reactions, amplification mixture (25 μL) contained 1 μL of primer mix, 2 μL of cDNA template, 12.5 μL of SYBR green Mix (Thermo Scientific Molecular Biology, MA, USA), and 9.5 μL of ddH₂O. Specific mRNA quantification was performed on ABI 7300 Real-Time PCR instrument (Applied Biosystems, CA, USA), according to the manufacturer's guidelines. The data were analyzed by use of ABI Prism 7300 Sequence Detection System software (Applied Biosystems, CA, USA). Expression levels of the mRNAs of interest were normalized to those of endogenous reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of mRNAs relative to GAPDH was calculated by the 2^−∆∆Ct method. All steps were performed under RNase-free conditions. Primers for JAK2, STAT3, Bcl-XL, Bax, and GAPDH in the reaction are listed in Table 1.

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.

Total RNA was extracted from the flavedo and juice sacs of Satsuma mandarin at different stages according to the method described by Ikoma et al. [54 (link)]. The total RNA was cleaned up using the RNeasy Mini Kit (Qiagen) with on-column DNase digestion. The reverse transcription (RT) reaction was performed with 2 μg of purified RNA and a random hexamer at 37 °C for 60 min using TaqMan Reverse Transcription Reagents (Applied Biosystems).
TaqMan MGB probes and sets of primers for CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C were designed with the Primer Express software (Additional file 4: Table S2). For the endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used. TaqMan real-time PCR was carried out with the TaqMan Universal PCR Master Mix (Applied Biosystems) using ABI PRISM 7300 (Applied Biosystems) according to the manufacturer’s instructions. Each reaction contained 900 nM of the primers, 250 nM of the TaqMan MGB Probe, and template cDNA. 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 with ABI PRISM 7300 Sequence Detection System Software (Applied Biosystems) and normalized with the results of 18S ribosomal RNA. Real-time quantitative RT-PCR was performed in three replicates for each sample.