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Cfx96 real time fluorescent quantitative pcr system

Manufactured by Bio-Rad
Sourced in United States

The CFX96 Real-time Fluorescent quantitative PCR system is a laboratory instrument designed for the amplification and detection of nucleic acid sequences using real-time quantitative PCR (qPCR) technology. The system enables the precise measurement and quantification of DNA or RNA targets in a sample.

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2 protocols using cfx96 real time fluorescent quantitative pcr system

1

Differential lncRNA Expression Analysis

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Gene expression was normalized using the fragments per kilobase of exon per million reads mapped (FPKM), which was calculated using Cuffdiff (v2.1.1) (22 (link)). Cuffdiff provides statistical routines for determining differential expression in the gene expression data using a model based on the contrary binomial distribution (22 (link)). Subsequently, differentially expressed lncRNAs at H2, H8, and H12 compared with H0 were filtered, and lncRNAs with a p-value < 0.05 were assigned as differentially expressed.
To validate the Illumina sequencing data, a total of six differentially expressed lncRNA were randomly selected for the qRT-PCR analysis. First, cDNA was synthetized using the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Japan). Then, specific primers were designed based on their sequences and EF1α was used as the internal control. The qRT-PCR was performed with the CFX96 Real-time Fluorescent quantitative PCR system (Bio-Rad, USA) using TB GreenTM Premix Ex TaqTM II (TaKaRa, Japan). The amplification cycle was as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 1 min, followed by a melting curve from 60 to 95°C. Data are shown as means ± SE for three replicates, and statistical analysis was performed using SPSS19.0.
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2

Carotenoid Pathway Gene Expression Analysis

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The peach reference genome was also used to establish a local database. Sequence probes were designed based on the reported genes related to carotenoid pathway, and BLASTP was compared with the local database to obtain the best matching results of genes related to carotenoid metabolism pathway. To verify the reliability of the sequencing data, 14 genes related to the carotenoid metabolic pathway were selected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis performed in CFX96 real-time fluorescent quantitative PCR system (Bio-Rad, https://www.bio-rad.com/). The operation steps refer to the SYBR® Premix Ex Taq manual, and the annealing temperature was 60°C. The relative expression levels of the genes were calculated using the 2-△△Ct method (Schmittgen and Livak, 2008 (link)). The sequences of oligonucleotide primers used in this study are presented in Supplementary Table S1.
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