Cfx96 real time pcr detection system

Manufactured by Roche

Sourced in United States, Switzerland

The CFX96 Real Time PCR Detection System is a real-time PCR instrument designed for gene expression analysis, SNP genotyping, and other quantitative PCR applications. The system utilizes a 96-well format and provides accurate and precise temperature control for reliable PCR results.

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Lab products found in correlation

Kapa sybr fast qpcr kit, by Roche (4 mentions) Transscript one step gdna removal and cdna synthesis supermix, by Transgene (4 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Axyprep multisource rna miniprep kit, by Corning (2 mentions) Mirvana extraction kit, by Thermo Fisher Scientific (1 mentions) Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions) Sybr green pcr master mix, by Roche (1 mentions) Revertra ace qpcr rt kit, by Toyobo (1 mentions) Taq mastermix, by CWBIO (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Plant rna isolation kit, by Tiangen Biotech (1 mentions) Lightcycler 480 sybr green master mix, by Roche (1 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Faststart universal sybr green master rox, by Roche (1 mentions) Rnaiso, by Takara Bio (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) Sybr premix ex taq, by Takara Bio (1 mentions) Maxima sybr green rox qpcr master mix 2, by Thermo Fisher Scientific (1 mentions) Lightcycler 480 probes master, by Roche (1 mentions)

12 protocols using cfx96 real time pcr detection system

Quantitative Gene and miRNA Expression Analysis

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Total RNA was isolated using TRIzol Reagent and the first-strand cDNA was synthesized using SuperScript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). QPCR was performed using SYBR® Green PCR Master Mix (Roche, Mannheim, Germany) on a CFX96™ Real-Time PCR Detection System. Relative mRNA levels were determined and standardized with a GAPDH internal control using the 2^−ΔΔCT method35 (link).
MiRNAs were extracted using the miRVana extraction kit (Ambion, Austin, TX, USA) and then reverse-transcribed and amplified using the microRNA reverse transcription and detection kit (Applied Biosystems, Inc. Foster City, CA). Primers for real-time PCR are all listed in Table 2. All results were normalized to U6 levels that were detected using the ABI miRNA U6 assay kit.

Cui Y., Han J., Xiao Z., Chen T., Wang B., Chen B., Liu S., Han S., Fang Y., Wei J., Wang X., Ma X, & Dai J. (2016). The miR-20-Rest-Wnt signaling axis regulates neural progenitor cell differentiation. Scientific Reports, 6, 23300.

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Quantifying Gene Expression Changes

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Total RNA was prepared from treated cells using TRIzol (Thermo Fisher Scientific). Briefly, after centrifugation, the aqueous layer was transferred to a fresh eppendorf tube, and isopropanol was added to precipitate total RNA. cDNA was generated from total RNA (1 µg) using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). qRT-PCR was performed with SYBR Green Master (Roche, Basel, Switzerland) on the CFX96 Real Time PCR Detection System (Roche). ACTB mRNA was used to normalize mRNA expression. The results are representative of at least three independent experiments. The sequences of the PCR primers used are the following: IL10-Forward: 5′-CCAGACATCAAGGCGCATGT-3′, Reverse: 5′-GATGCCTTTCTCTTGGAGCTTATT-3′; TGF-β- Forward: 5′-GCAACAATTCCTGGCGATACC-3′, Reverse: 5′-ATTTCCCCTCCACGGCTCAA-3′; TNF-α- Forward: 5′- GAGGCCAAGCCCTGGTATG-3′, Reverse: 5′-CGGGCCGATTGATCTCAGC-3′; BST2- Forward: 5′-ATGGAAGACGGGGATAAGCG-3′, Reverse: 5′-GGAGATGGGTGACATTGCGA-3′; ACTB- Forward: 5′-CATGTACGTTGCTATCCAGGC-3′, Reverse: 5′-CTCCTTAATGTCACGCACGAT-3′.

Kong Y., Xue Z., Wang H., Cui G., Chen A., Liu J., Wang J., Li X, & Huang B. (2022). Identification of BST2 Contributing to the Development of Glioblastoma Based on Bioinformatics Analysis. Frontiers in Genetics, 13, 890174.

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Quantitative Real-Time RT-PCR Analysis

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Total RNA was isolated from different organs using a plant RNA isolation kit (Tiangen). The RNA sample (3 μg) was used for complementary DNA synthesis with the SuperScript III (Invitrogen) according to the manufacturer's instructions. RT–PCR was performed with Taq Master Mix (CWBIO) using ACTIN7 as a control. Quantitative real-time RT–PCR analysis was performed with the Bio-Rad CFX96 real-time PCR detection system using the LightCycler 480 SYBR Green Master Mix (Roche). ACTIN2, TUB2, UBQ10, GAPDH or EF1A mRNAs were used as internal controls. Relative amounts of mRNA were calculated using the Cycle threshold (Ct) method. Ct values correspond to the cycle number at which the fluorescence resulting from enrichment of the PCR product reaches significant levels above the background fluorescence. The ΔCt was determined by subtracting the Ct values of ACTIN2, TUB2, UBQ10, GAPDH or EF1A from the SAP Ct value. The ratios were calculated as being equal to 2^−ΔCt. PCR reactions were performed in triplicate for each sample. The primers used for RT–PCR and quantitative real-time RT–PCR are listed in Supplementary Table 2.

Wang Z., Li N., Jiang S., Gonzalez N., Huang X., Wang Y., Inzé D, & Li Y. (2016). SCFSAP controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana. Nature Communications, 7, 11192.

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Comparative Transcriptomics of ALV-J-Infected DF-1 Cells

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IP and input RNA from control and ALV-J-infected DF-1 cells were converted to cDNA using RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher) following the manufacturer's instructions. qPCR was performed on a BIO-RAD CFX96 Real-Time PCR Detection System using Roche FastStart Universal SYBR Green Master (Rox) (Roche, Switzerland) and specific primer sets (Supplementary Table 1). The qPCR results with 3 independent replications were analyzed with relative expression of the Ct (threshold cycle) value using the 2^−△△Ct method. Statistical analysis were performed using Graph Prism 9.1.0 software with student's t-test employed to evaluate the groups’ differences. A probability values (p value) less than 0.05 was considered to be statistically significant.

Ji J., Xu S., Xu X., Man Y., Yao L., Xie Q, & Bi Y. (2024). Transcriptome-wide N6-methyladenosine modification and microRNA jointly regulate the infection of avian leukosis virus subgroup J in vitro. Poultry Science, 103(6), 103671.

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Quantitative Real-Time PCR for Gene Expression

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Total RNA was extracted from cells using RNAiso (Takara, Japan) according to the manufacturer's protocol. Reverse transcription was conducted using the PrimeScript™ RT reagent kit (Takara, Japan). qRT-PCR was performed with SYBR premix Ex Taq (Takara, Japan) on the CFX96 Real Time PCR Detection System (Roche 480II; Berlin, Germany). GAPDH mRNA was used to normalize mRNA expression, and the results are representative of at least three independent experiments. The sequences of the primers used are shown in Supplementary Table S1.

Han M., Xu R., Wang S., Yang N., Ni S., Zhang Q., Xu Y., Zhang X., Zhang C., Wei Y., Ji J., Huang B., Zhang D., Chen A., Li W., Bjerkvig R., Li X, & Wang J. (2018). Six-Transmembrane Epithelial Antigen of Prostate 3 Predicts Poor Prognosis and Promotes Glioblastoma Growth and Invasion. Neoplasia (New York, N.Y.), 20(6), 543-554.

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T Cell Transcriptome and qPCR Analysis

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RNA was extracted from purified CD4+ T_N and T_CM cells and from 24ST1NLESG cells using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Transcriptome analysis for the CD4+ T_N and T_CM cells was carried out by RNA sequencing (RNA-Seq; GENEWIZ, Azenta Life Sciences, South Plainfield, NJ, USA). The RNA from 24ST1NLESG cells was DNase-treated with RNase-Free DNase (Qiagen, Valencia, CA, USA) and reverse transcribed using random primers with the AffinityScript multiple temperature reverse transcriptase (Agilent, Wilmington, DE, USA). Quantitative PCR (qPCR) was performed using a Bio-Rad CFX96 real-time PCR detection system, using 2× Lightcycler 480 probes master (Roche, Indianapolis, IN, USA) for IPO8 and Maxima SYBR Green/ROX qPCR Master Mix (2×) (Thermofisher, Waltham, MA, USA) for PAK, using primers described in Supplementary Table S1. Quantification for each qPCR reaction was assessed by the 2^−ΔCT algorithm, relative to IPO8 expression.

Vargas B., Boslett J., Yates N, & Sluis-Cremer N. (2023). Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency. Biomolecules, 13(1), 100.

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Quantifying Gene and miRNA Expression

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Total RNA was extracted from 200 mg tissue from LA, LV, and AA using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions [9 (link)]. cDNA synthesis for elastin quantification was performed using a first-strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) from 1-5 μg of total RNA; amount of total RNA was the same within the pair transcript-gene of interest. MiRNA quantification was performed from 2.5 μg of total RNA using the NCode^™ miRNA first-strand cDNA synthesis kit (Life technologies, cat #45-6612) with GoTaq® qPCR Master Mix (Promega A6002, Madison, USA) [18 (link), 45 (link)]. qPCR with 10 ng/reaction was performed on the CFX96^™ Real-Time PCR Detection System using SYBR Green supermix (Kapa Bio systems Inc. Woburn, MA, USA). Quantitative PCR was performed in duplicates with sequence-specific oligonucleotides, that were custom-synthesized [59 (link)] (Thermo Fisher Scientific Inc, Waltham, MA, USA and Invitrogen, Carlsbad, CA, USA) and also commercially available (Roche, Applied sciences, Indianapolis, IN, USA) (Table 2) [16 (link)]. Data were collected on a LightCycler^® 480 (Roche, Applied Sciences, Indianapolis, IN, USA) for ELN expression. Gene expressions were compared to suitable reference genes (i.e., β-actin, s-19, and UC6) and normalized using the 2^−ΔΔC_T method.

SCHLABRITZ – LOUTSEVITCH N., APOSTOLAKIS-KYRUS K., KRUTILINA R., HUBBARD G., KOCAK M., JANJETOVIC Z., SATHANANDAM S., SLOMINSKI A., MARI G, & DICK E J.r. (2016). PREGNANCY-DRIVEN CARDIOVASCULAR MATERNAL MIR-29 PLASTICITY IN OBESITY. Journal of medical primatology, 45(6), 297-303.

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Quantitative RT-PCR for mRNA and microRNA

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RT-qPCR was used to detect the mRNA and microRNA expression in total RNAs extracted from cultured cells using TRIZOL reagent (Invitrogen). Reverse transcription of the total RNAs to cDNA using reverse transcriptase from TOYOBO (Osaka, Japan). Actin was used as an internal control. For miRNA detection, a stem-loop RT primer was designed for microRNA hybridization and RNU6B was used for normalization. PCR was performed in a Bio-Rad CFX96 real-time PCR detection system using SyBr Green reagents from KAPA Biosystems (Wilmington, MA, USA). PCR primers used are described in the Additional file 1: Table S3.

Jang T.H., Huang W.C., Tung S.L., Lin S.C., Chen P.M., Cho C.Y., Yang Y.Y., Yen T.C., Lo G.H., Chuang S.E, & Wang L.H. (2022). MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/β-catenin pathway. Journal of Biomedical Science, 29, 42.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA from inflorescences of four plants at 8 days after bolting was extracted using
the AxyPrep Multisource RNA MiniPrep kit (Axygen, Tewksbury, MA, USA). First-strand cDNA was
synthesized with 2 μg total RNA by TransScript One-step gDNA Removal and cDNA
synthesis SuperMix (TransGen, Beijing, China) using anchored oligo-dT primers according to the
manufacturer's instructions. Quantitative real-time PCR (qRT–PCR) was performed on a
Bio-Rad CFX96 real-time PCR detection system using KAPA SYBR FAST qPCR kit (KAPA Biosystems,
Beijing, China). TUB6 (AT5G12250) was chosen to normalize the relative expression
as it has been shown to be a superior reference gene for qRT–PCR analysis (Han
et al, 2014 (link)). Gene-specific primers
(Supplementary Table S12) were used to amplify each gene, and two independent biological
experiments, each run in triplicate, were applied for each mutant or transgenic plant.

Tian C., Zhang X., He J., Yu H., Wang Y., Shi B., Han Y., Wang G., Feng X., Zhang C., Wang J., Qi J., Yu R, & Jiao Y. (2014). An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation. Molecular Systems Biology, 10(10), 1-2.

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RNA Extraction and RT-qPCR Analysis

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Total RNA was extracted from inflorescences or leaves using the RNeasy Micro kit (Qiagen) according to the
manufacturer’s instructions. For Dex/CHX treatment experiments, transgenic shoot apexes were treated by 10 μM
Dex alone or with 10 μM CHX for 4 hr. The first strand of cDNA was synthesized using TransScript One-Step gDNA Removal
and cDNA synthesis SuperMix (TransGen), and then used as templates for reverse transcription quantitative PCR (RT-qPCR).
RT-qPCR was performed through the Bio-Rad CFX96 real-time PCR detection system using KAPA SYBR FAST qPCR kit (KAPA
Biosystems). The relative expression levels of target genes were normalized to ACTIN2(At3g18780) levels. All primers used in RT-qPCR were listed in Table S1.

Guan C., Wu B., Yu T., Wang Q., Krogan N.T., Liu X, & Jiao Y. (2017). Spatial auxin signaling controls leaf flattening in Arabidopsis. Current biology : CB, 27(19), 2940-2950.e4.

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