Total RNA was extracted from liver tissues with a standard Trizol RNA extraction procedure. RNA quality was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent, USA). Sequencing libraries were generated using the NEBNextR Ultra RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA). The size of the library insert was tested using the Agilent Bioanalyzer 2100 system (Agilent, USA). The Bio-RAD CFX 96 fluorescent quantitative PCR instrument and Bio-RAD KIT iQ SYBR GRN were used to perform Q-PCR for accurate quantification of the effective concentration of the library (effective concentration of the library > 10 nM). After cluster generation, the libraries were sequenced by the Illumina NovaSeq 6000 S4 platform with paired-end reads by Annoroad Gene Technology (Beijing, China). Differential gene expression (DEGs) analysis was performed using the DESeq2R package version 1.16.3. and the DEGs were selected with |LogFC|> 1 and FDR < 0.05.
Cfx 96 fluorescent quantitative pcr instrument
Manufactured by Bio-Rad
Sourced in United States
The CFX 96 is a fluorescent quantitative PCR (qPCR) instrument designed for real-time detection and quantification of nucleic acid sequences. It features a 96-well block and supports a wide range of fluorescent dyes and probes for gene expression analysis, pathogen detection, and other applications. The CFX 96 provides precise temperature control and sensitive fluorescence detection to enable accurate and reproducible qPCR results.
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Lab products found in correlation
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Novaseq 6000 s4 platform, by Illumina (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Kit iq sybr grn, by Bio-Rad (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Sureselectxt target enrichment system, by Agilent Technologies (1 mentions)
Qubit 3, by Agilent Technologies (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Transstart probe qpcr supermix, by Transgene (1 mentions)
4 protocols using cfx 96 fluorescent quantitative pcr instrument
1
Liver Transcriptome Profiling by RNA-seq
2
High-Throughput Sequencing Library Preparation Protocol
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The Agilent SureSelectXT Target Enrichment System (G7530-90000) was used for small fragment library preparation, including the following: end repair, A-tailing, adapter ligation, purification, and amplification. After construction, the quality of the sequencing libraries was assessed. Initial quantitative analyses were performed using Qubit 3.0, and the sizes of the sequencing libraries were measured by Agilent 2100. Using Bio–Rad's CFX 96 fluorescent quantitative PCR instrument and Bio–Rad's iQ SYBR Green Kit for the Q-PCR test, the concentration of each library was accurately quantified (>10 nM) to ensure quality. Each sample was 2 × 150 bp double-end sequenced with an Illumina HiSeq 4000 (Illumina, USA), and approximately 6G of raw sequencing data was output (2×). The flowchart of the experiment was shown in Figure 1 .Figure 1 ![]()
Flowchart of the experiment.
3
Sheep Skin Development and Gene Expression
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Fetal skin tissue at 65d, 85d, 105d, 135d, and 7d and 30d after birth (labeled as G1, G2, G3, G4, G5, and G6, respectively) were used, and three samples were collected at each stage. According to the sequences of sheep DNMT1, DNMT3a, DNMT3b, and EDN1 provided by the NCBI, the reference gene was GAPDH, and Primer Premier 5.0 software was used to design the quantitative primers. The cDNA template was used for qRT-PCR amplification. The total system was 25μl, including 1μl cDNA, and each sample had three replicates. The amplification reaction was carried out in Bio-Rad CFX96 fluorescent quantitative PCR instrument.
The gene expressions of WNT2 of the skin, heart, liver, spleen, lung, kidney, and muscle tissue of left shoulder of the lambs at 135d (hair follicle maturation period) were identified. According to the sequences of sheep WNT2, the reference gene was GAPDH (Kang et al., 2020 (link)). The amplification procedures and systems were same as above.
The gene expressions of WNT2 of the skin, heart, liver, spleen, lung, kidney, and muscle tissue of left shoulder of the lambs at 135d (hair follicle maturation period) were identified. According to the sequences of sheep WNT2, the reference gene was GAPDH (Kang et al., 2020 (link)). The amplification procedures and systems were same as above.
4
Real-Time PCR Genotyping Assay
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Primer sequences and their Taqman probe sequences at SNP site (Table II ) designed by Oligo6.0 were used. Primer synthesis was accomplished by Sangon Biotech Co., Ltd. (Shanghai, China). DNA solution (1 µl) and 1.2 µl prepared primer solution (including 0.4 µl upstream primer, 0.4 µl downstream primer and 0.4 µl probe primer) were added to 17.8 µl pre-prepared TransStart Probe qPCR SuperMix (Beijing TransGen Biotech Co., Ltd., Beijing, China), slightly shaken to mix, and placed into a CFX96 fluorescent quantitative PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction conditions: i) 94°C for 3 min, for 1 cycle; ii) 94°C for 15 sec and 60°C for 60 sec, for 42 cycles. After each cycle, the fluorescence value was read once. The experimental results were generated by built-in software of the instrument. Three replicate wells were made for each sample, diethyl pyrocarbonate (DEPC) water was used as negative control, and positive plasmid containing the sequence (synthesized by Sangon Biotech) was used as positive control. Determination of genotypes: The wild homozygous genotype was near the FAM abscissa, the mutant homozygous genotype was near the VIC ordinate, and the heterozygous genotype was near the 45° line.
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