Cfx96 touch real time pcr c1000 thermal cycler system

Total RNA from the fruit samples was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) and treated with a TURBO DNA-free^™ kit (Ambion, Austin, TX, USA) to remove DNA contamination following the manufacturers’ protocol. Purified RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Carlsbad, Inc., Carlsbad, CA, USA) and RNA integrity was evaluated using 1% agarose gel electrophoresis. First-strand complementary DNA synthesis was conducted using a reverse transcription kit containing a PrimeScript^TM RT reagent kit and gDNA Eraser (Perfect Real Time) (Takara, Dalian, China).
The detailed sequences of primers used are listed in Table 1; moreover, qRT-PCR was performed on a CFX96 Touch Real-Time PCR C1000 Thermal Cycler system (Bio-Rad, Hercules, CA, USA) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer’s instructions. The internal reference genes for the normalization of relative gene expression were obtained from Fu et al. [22 (link)]. Three technical replicates were performed for each biological replicate.

Total RNA from the fruit samples described above was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) and treated with TURBO DNA-free^™ kit (Ambion, Austin, TX, USA) to remove DNA contamination, following the manufacturer’s protocol. Purified RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was evaluated using 1% agarose gel electrophoresis. First-strand complementary DNA synthesis was conducted using a reverse transcription kit containing a PrimeScript^TM RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China).
Genes related to OA metabolism were selected according to the loquat whole genome sequence [22 (link)]. The internal reference genes for normalization of relative gene expression were obtained from [19 (link)]. PEPC2, NAD-MDH, NADP-ME2, VHA-A, and VHP1 were obtained from [23 (link)]. ALTMs were obtained from [24 (link)]. The detailed sequences of primers used are listed in Supplementary Table S1.
qRT-PCR was performed on a CFX96 Touch Real-Time PCR C1000 Thermal Cycler system (Bio-Rad, Hercules, CA, USA) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies Inc., Santa Clara, CA, USA), following the manufacturer’s recommendations. For each biological replicate, three technical replicates of each PCR were performed.

The specific primers targeting the genes associated with carotenoid biosynthesis can be found in Table 3 and were procured from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), The PsActin gene served as the internal reference gene. The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the subsequent purification of mRNA was carried out using a TURBO DNA-free kit (Ambion, Austin, TX, USA). Complementary DNA synthesis was performed using a PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). The qRT-PCR analysis was conducted using a CFX96 Touch Real-Time PCR C1000 Thermal Cycler system (Bio-Rad, Hercules, CA, USA) and an SYBR^® Premix Ex Tag^™ II (Tli RNaseH Plus) (Takana, Dalian, China), according to the manufacturer’s instructions. The amplification programs consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 58 °C for 30 s. Each biological replicate was subjected to triplicate PCR amplification. The relative expression levels of the target genes were normalized to those of the internal reference gene (PsActin) using the 2^−(ΔΔCt) method.