The cDNA was synthesized using the total RNA of the curved stem in lazy1 and the parallel erect part in WT at 10 days after germination (bending stage) for the detection of qRT-PCR. To explain the phenotype of lazy1 and verify the reliability of RNA-seq, a total of 25 genes were selected to perform qRT-PCR using SoFast™ EvaGreen® Supermix and CFX96™ (Takara, Dalian, China). Transcript levels were normalized against Actin. The cDNA was amplified under the following cycling conditions: (1) one cycle of 95°C for 30 s, (2) 40 cycles of 95°C for 5 s, 60°C for 30 s, and (3) melt-curve from 65 to 95°C by 5 s per step with a 0.5°C increment. The sequences of gene-specific primers are listed in Supplementary Table 1 .
Cfx96
Manufactured by Takara Bio
Sourced in China
The CFX96 is a real-time PCR detection system. It is designed to perform quantitative and qualitative PCR experiments with high accuracy and precision.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Sybr green kit, by Takara Bio (2 mentions)
Sybr green premix ex taq 2, by Takara Bio (2 mentions)
Primescript rt reagent kit, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Fastquant rt kit, by Tiangen Biotech (1 mentions)
Novaseq sequencer, by Illumina (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Easyspin plus rna extraction kit, by Aidlab (1 mentions)
Ssofast evagreen supermix, by Takara Bio (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Takara Bio (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
11 protocols using cfx96
1
qRT-PCR Analysis of Lazy1 Gene
2
Wheat Transcriptional Response to Powdery Mildew
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The wheat leaf samples of YAV249 were collected at 0, 2, 4, 8, 12, 24, 48 and 72 hours post inoculation (hpi) after inoculating Bgt isolate E09 and with three biological replicates. Total RNA was extracted using RNAiso Plus (TaKaRa, Shiga, Japan). For each sample, 2 μg of RNA was used for reverse transcription with a FastQuant RT Kit (Tiangen, Beijing, China). The transcript levels were detected by qRT-PCR on Bio-Rad CFX 96 with TB Green Premix Ex Taq™ II (TaKaRa, Shiga, Japan). The procedure included an initial step at 95°C for 30 s followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 10 s. The expression levels of target genes were normalized to that of TaActin. All qRT-PCR assays were performed in three independent replications.
3
Transcriptomic Analysis of FGFR4 Knockdown in Breast Cancer
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TRIzol reagent (Invitrogen) was used to extract total RNA from cells and tissues. After reverse transcription was completed, the RNA expression level was examined by qPCR in triplicate on a Bio-Rad CFX96 using the SYBR Green kit (Takara, RR420A). Primer sequences are shown in Supplementary Table 5 . Total RNA was extracted from sg-NC or sg-FGFR4 group of rSKBR3 breast cancer cells (2 × 106) using TRIzol reagent (Invitrogen) for sequencing analysis. After the assessment of RNA integrity, the mRNA library was generated and sequenced by CapitalBio Technology (Beijing, China). The NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used to construct the libraries for sequencing. All next-generation sequencing experiments were run on an Illumina NovaSeq sequencer (Illumina). Differentially expressed genes were subsequently screened out and identified with the criterion of |log2FC|> 1 and FDR < 0.05. DAVID tool was used to conduct Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Gene set enrichment analysis (GSEA) was applied to identify the significantly enriched pathways between the two groups using the GSEA 4.1.0 desktop application.
4
Quantifying lncRNA and miRNA Expression in Breast Cancer
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RNA samples were isolated from BC tissues and cells by using TRIzol reagent (Invitrogen, Carlsbad, CA). Then, the RNAs were reverse-transcribed into complementary according to the PrimeScript RT reagent kit (Invitrogen, Shanghai, China). Bio-Rad CFX96 and SYBR Green Premix Ex Taq II (Takara, Dalian, China) were used for qRT-PCR. LightCycler 96 thermocycler was used to investigate the relative expression of NR2F1-AS1 and miR-641. The expression of NR2F1-AS1 was normalized to GAPDH, while that of miR-641 was standardized to U6. The primer sequences were listed as follows: NR2F1-AS1, forward: 5′-CGCGAGGGCGTAAAAGTTTG-3′ and reverse: 5′-AGCGTGGCCATTTACTTCCA-3′; miR-641, forward: 5′-GGCTGGGTGAAAGGAAGGAA-3′ and reverse: 5′-GAGGCTCAGGGGTAAGAGGA-3′; GAPDH, forward: 5′-CCTGACCTGCGTGTGGACT-3′ and reverse: 5′-GCTGTGGATGGGGAGGTGTC-3′; and U6, forward: 5′-CTCGCTTCGGCAGCACA-3′ and reverse: 5′-TGGTGTCGTGGAGTCG-3′.
5
Quantitative Gene Expression Analysis
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Total RNA was extracted by Trizol reagent (Sigma, T9424) and transformed into cDNA by the reverse transcription kit (Invitrogen, 18080051). qPCR analysis was repeated three times on Bio-Rad CFX96 using SYBR premix ex Taq (TaKaRa, RR820). All qPCR data were standardized by GAPDH. The primer information is shown in Supplementary Table S2 .
6
Validating RNA-seq Data by qRT-PCR
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Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the accuracy of the RNA-seq. Ten genes were randomly selected for verification. TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA using a reverse transcription kit (Takara Co., Ltd., Dalian, China). qRT-PCR was performed using a Bio-Rad CFX96 with a SYBR Premix ExTaq™ kit (Takara Bio, Inc.) according to the manufacturer’s instructions. Primers were synthesized by Shanghai Sangon Co., Ltd. (Shanghai, China), and these sequences can be found in Supplementary Table 1. Gene expression was quantified relative to the expression of ACTB using the comparative cycle threshold (ΔCT) method. Each sample was run in triplicate.
7
Quantitative RT-PCR Analysis of Tomato Transcripts
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Total RNA was extracted from tomato seedlings using the TRIzol reagent (Invitrogen). cDNA was made from 2 μg of total RNA with SuperScript III reverse transcriptase (Invitrogen) and quantified on a Bio-Rad CFX96 with the SYBR Green kit (Takara). Tomato EF1a was used as an internal control. Statistical significance was evaluated by Student’s t-test. Primers are listed in Supplementary Data Table S6 .
8
Quantitative Analysis of DoUGP Expression
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Total RNA from the wild-type and three independent overexpression lines was extracted using 100 μg of 12-month-old plants by using an EASYspin Plus RNA extraction kit (Aidlab, Beijing, China), and cDNA was synthesized using a first-strand cDNA synthesis kit (Thermo, Waltham, MA, United States). Quantitative real-time PCR (qRT-PCR) was performed using SsoFast EvaGreen Supermix and CFX96 (TaKara, Dalian, China) to evaluate the expression levels of DoUGP and its metabolic upstream and downstream genes in the wild-type and transgenic plants. Transcript levels were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The cDNA was amplified under the following cycling conditions: (1) one cycle of 95°C for 30 s; (2) 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 15 s; and (3) melt-curve from 65°C to 95°C by 5 s per step with a 0.5°C increment. The sequences of gene-specific primers used for the amplifications are listed in Supplementary Table S1 . Results are presented as mean ± standard error calculated from three biological replicates.
9
Quantitative RT-PCR for Gene Expression
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First-strand cDNA was generated from total RNA using the RevertAid First Strand cDNA Synthesis Kit following the manufacturer’s protocol (Thermo Scientific). qRT-PCR was performed on a Bio-Rad platform (CFX96) using a SYBR Green detection chemistry kit (SYBR® Premix ExTaq™, TaKaRa). Each 13 μL mixture contained 6.25 μL of SYBR Green Supermix (TaKaRa), 1.0 μL of cDNA, 0.375 μL of each primer, and distilled water. The program used for qRT-PCR was as follows: 95°C for 30 s; followed by 35 cycles of denaturation at 95°C for 5 s and annealing at 62°C for 30 s. The housekeeping genes ZmActin3 and AtActin2 were each used as an internal control. All primers used for qRT-PCR are given in S2 Table . The 2-ΔCT method was used to estimate the fold change. The data were analyzed using the Bio-Rad CFX Manager software. Three biological replicates with three technical replicates were used for each reaction.
10
Quantitative Real-Time PCR Analysis of lncRNA CASC9 and miR-193a-5p
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TRIzol (Invitrogen, Carlsbad, CA, USA) was employed to extract the total RNA from tissues and cells. In compliance with the supplier’s instructions, total RNA was reversely transcribed into cDNA with PrimeScript RT Reagent Kit (Invitrogen, Shanghai, China). Bio-Rad CFX96 and SYBR Green Premix Ex Taq II (Takara, Dalian, China) were adopted for RT-PCR, which was carried out according to the manufacturer’s regulations. GAPDH and U6 were used as reference genes, and 2(-ΔΔCt) method was used for statistics. The specific primer sequence information is shown in Table 1 .
The Sequence of PCR Primers Used
| Name | Primer Sequences |
|---|---|
| lncRNA CASC9 | Forward: 5ʹ-TTGGTCAGCCACATTCATGGT-3’ |
| Reverse: 5ʹ-AGTGCCAATGACTCTCCAGC-3’ | |
| miR-193a-5p | Forward: 5′-TGGGTCTTTGCGGGC-3′ |
| Reverse: 5′-GAATACCTCGGACCCTGC-3′ | |
| U6 | Forward: 5′-CTCGCTTCGGCAGCACATA-3′ |
| Reverse: 5′- AACGATTCACGAATTTGCGT-3 | |
| ERBB2 | Forward: 5′-CCAGCCTTCGACAACCTCTATT-3′ |
| Reverse: 5′- TGCCGTAGGTGTCCCTTTG-3 | |
| GAPDH | Forward: 5′-GGGAGCCAAAAGGGTCATCA-3′ |
| Reverse: 5′-TGATGGCATGGACTGTGGTC-3′ |
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