Cfx96 real time pcr detection system

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The CFX96 Real-Time PCR Detection System is a thermal cycler designed for real-time PCR analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time using fluorescent dyes or probes.

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4 278 protocols using cfx96 real time pcr detection system

Quantitative Analysis of Inflammatory Cytokines

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Total RNA was isolated from midbrain by using RNAiso Plus (Takara) following the standard protocol. For quality control, RNA purity was quantified by the NanoDrop 2000 spectrophotometer (Thermo Scientific). Total RNA (1 μg) was reverse-transcribed to cDNA by using a Reverse Transcription Kit (Takara). Real-time quantitative PCR was performed with SYBR® Premix Ex TaqTM II (Takara) and the CFX96TM Real-Time PCR Detection System (Bio-Rad). The following primers were designed and synthesized by Life Technologies: IL-1β, forward, GAA ATG CCA CCT TTT GAC AGT G, and reverse TGG ATG CTC TCA TCA GGA CAG; IL6, forward TAG TCC TTC CTA CCC CAA TTT CC, and reverse TTG GTC CTT AGC CAC TCC TTC; TNFα, forward, CAG GCG GTG CCT ATG TCT C, and reverse CGA TCA CCC CGA AGT TCA GTA G; Actb, forward, GGC TGT ATT CCC CTC CAT CG, and reverse CCA GTT GGT AAC AAT GCC ATG T. GAPDH, forward, AGG TCG GTG TGA ACG GAT TTG, and reverse TGT AGA CCA TGT AGT TGA GGT CA.
All samples were analyzed and normalized with the expression levels of two housekeeping genes (β-actin and GAPDH). The mRNA levels were analyzed and quantified with the 2−ΔΔCt method by CFX Manager Software provided by the CFX96TM Real-Time PCR Detection System (BioRad Laboratories, Inc.).

Mo Y., Xu E., Wei R., Le B., Song L., Li D., Chen Y., Ji X., Fang S., Shen J., Yang C, & Wang Q. (2018). Bushen-Yizhi Formula Alleviates Neuroinflammation via Inhibiting NLRP3 Inflammasome Activation in a Mouse Model of Parkinson's Disease. Evidence-based Complementary and Alternative Medicine : eCAM, 2018, 3571604.

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Comprehensive mRNA and miRNA Expression Analysis

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For mRNA expression analyses (RT‐PCR), total RNA were isolated using the RNA Fast 200 isolation kit (220010, Feijie) and reversely transcribed using the cDNA Reverse Transcription Premix (11123, Yeasen) according to the manufacturer’s instructions. A total of 1 μL of cDNA was used for quantitative (qPCR) analyses. qPCR mixtures were prepared using the SYBR Green qPCR Mix (11201, Yeasen). qPCR analyses were performed with the Bio‐Rad CFX96TM Real‐Time PCR Detection System. mRNA expression was normalized to GAPDH and determined using the ΔΔCt method. The primers used in this study are shown in Table S2.
For miRNA expression analysis, miRNA were isolated with the miRcute miRNA Isolation Kit (DP501, TIANGEN). All miRNA were polyadenylated by poly(A) polymerase and converted into cDNA with the miDETECT A Track miRNA qRT‐PCR Starker Kit (C10712, RIB‐BIO). qPCR mixtures were prepared, and qPCR analyses were performed with the Bio‐Rad CFX96TM Real‐Time PCR Detection System. U6 was used as an internal reference to normalize miRNA expression. All primers used were obtained from RIB‐BIO. Two microDNA chips of lung cancer tissues (#micDNA‐HLugC030PT01 and #micDNA‐HLugA030PG01, each containing 15 pairs of lung cancer samples, respectively) used for analyzing miR‐1227‐5p expression were purchased from Shanghai OUTDO Biotech.

Yang L., Wang X., Jiao X., Tian B., Zhang M., Zhou C., Wang R., Chen H., Wang B., Li J., Liu J., Zhang G, & Liu P. (2020). Suppressor of Ty 16 promotes lung cancer malignancy and is negatively regulated by miR‐1227‐5p. Cancer Science, 111(11), 4075-4087.

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Bacterial DNA Quantification from Ileal and Cecal Digesta

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Bacterial DNA in the ileal and cecal digesta was extracted by using the Stool DNA Kit (Omega Bio-tek) according to the manufacturer’s instruction. The microbial real-time quantitative PCR was determined as described previously [34 (link)]. Briefly, the number of total bacteria was analyzed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA), and the number of Lactobacillus, E. coli and Bifidobacterium was analyzed by real-time quantitative PCR using PrimerScriptTM PCR kit (Perfect Real Time; TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) as previously described (Chen et al., 2013). All primers and probes were purchased by TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China), which was listed in Table 3. For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (2013) [34 (link)]. In addition, bacterial copies were transformed (log₁₀) before statistical analysis.

Mao X., Gu C., Hu H., Tang J., Chen D., Yu B., He J., Yu J., Luo J, & Tian G. (2016). Dietary Lactobacillus rhamnosus GG Supplementation Improves the Mucosal Barrier Function in the Intestine of Weaned Piglets Challenged by Porcine Rotavirus. PLoS ONE, 11(1), e0146312.

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Quantitative analysis of gene expression

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For the assay of relative transcription levels of targe genes, sample preparation and RNA extraction were performed using the method described previously [7 (link)]. Quantitative PCR was carried out with SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) using a CFX96 real-time PCR detection system (Bio-Rad). The PCR reaction mixture (with three replicates) included 75 ng of template RNA, 0.4 μL of each primer (10 μM), 10 μL of RNA-direct SYBR^® Green Realtime PCR Master Mix, and 8.2 μL of H₂O. Negative controls contained an equal volume of water instead of RNA. Actin gene (MYCTH_2314852) was used as an internal control. The relative transcript level of each gene was calculated by the 2^−ΔΔCt method.
For copy number assay of genes ectopically inserted into M. thermophila genome, fungal genomic DNA was extracted from transformants as described previously and used as the template for RT-qPCR. Quantitative PCR was carried out with SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) with a CFX96 real-time PCR detection system (Bio-Rad), according to the manufacturer’s instructions. The oligonucleotides of the primers for each gene were optimized to obtain amplification efficiency between 95 and 105% and only one melting temperature on the melting curve.
The primers used for RT-qPCR are listed in Additional file 1.

Li J., Gu S., Zhao Z., Chen B., Liu Q., Sun T., Sun W, & Tian C. (2019). Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila. Fungal Biology and Biotechnology, 6, 21.

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Caecal Microbiome Quantification Protocol

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Microbial genomic DNA in the caecal digesta was extracted by using the Stool DNA Kit (Omega Bio-Tech) according to the manufacturer's instruction. The microbial real-time quantitative PCR was determined as described previously (20) . All primers and probes for total bacteria, Escherichia coli, Lactobacillus, Bifidobacterium and Bacillus (21) (Table 3) were commercially synthesised from TaKaRa Biotechnology (Dalian) Co. Ltd. Briefly, the number of total bacteria was analysed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co. Ltd) and CFX-96 real-time PCR detection system (BioRad Laboratories), and the numbers of Bacillus, Lactobacillus, E. coli and Bifidobacterium were analysed by real-time quantitative PCR using PrimerScript TM PCR kit (perfect real time; TaKaRa Biotechnology (Dalian) Co. Ltd) and CFX-96 real-time PCR detection system (Bio-Rad Laboratories) as previously described (20) . For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (20) . In addition, bacterial copies were transformed (log 10 ) before statistical analysis.

Wang W., Chen D., Yu B., Huang Z., Mao X., Zheng P., Luo Y., Yu J., Luo J., Yan H, & He J. (2020). Effects of dietary inulin supplementation on growth performance, intestinal barrier integrity and microbial populations in weaned pigs. The British journal of nutrition, 124(3).

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Quantitative RNA Expression Analysis

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Total RNA was extracted using TRIzol Reagent (Invitrogen, Garlsbad, CA, USA). The purity of RNA was assessed by Nano-Drop 2000 (Thermo, USA) and only highly pure RNAs (1.7< A260/A280 <2.2) were usable. For mRNA analysis, 1 μg of total RNA from each sample was used in the reverse transcription reaction using a Revert Aid First Strand cDNA Synthesis Kit (Fermentas, USA) and Real-time qPCR was performed with a SYBR Premix Ex Taq II (Takara, China) and a CFX-96 Real-time PCR Detection System (Bio-Rad, USA). GAPDH was used as control. For miRNA analysis, 1.5 μg of total RNA from each sample was used to sythesize complementary DNA using TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and Real-time qPCR was performed with a TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA) and a CFX-96 Real-time PCR Detection System (Bio-Rad, USA). U6 was used as control. All the data were analyzed using the 2 ^−ΔΔCt method and all experiments were carried out in triplicate. The specific primers (Genomics Institute, China) were shown in Table 2.

Wang Z., Wang W., Huang K., Wang Y., Li J, & Yang X. (2017). MicroRNA-34a inhibits cells proliferation and invasion by downregulating Notch1 in endometrial cancer. Oncotarget, 8(67), 111258-111270.

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Quantifying miRNA and mRNA Expression

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For miRNA expression, qPCR was performed using TaqMan miRNA Assays (Applied Biosystems): mmu-miR-5099, mmu-miR-5102, hsa-miR-486, hsa-miR-214, hsa-199a-5p and cel-miR-39. The small nuclear U6 RNA (RNU6) was used as endogenous control for human samples, and hsa-let-7a and snoRNA202 for mouse samples. cDNA was synthesized using TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed with TaqMan probes on a CFX96 real-time PCR detection system (Bio-Rad) as follows: 10 min at 95 °C, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative miRNA expression levels were compared using 2^−ΔΔCt method. Quantitative miRNA data were analyzed using CFX96 real-time PCR detection system (Bio-Rad).
For mRNA expression of potential miR-486 targets, MEFs treated with 10X P3F-C2C12 exosomes or Ctrl-C2C12 exosomes for 48 h were lysed and total RNA was reverse transcribed using QuantiTect Reverse Transcription kit (Qiagen) followed by qPCR with QuantiFast SYBR Green PCR (Qiagen) for 40 cycles using primers listed in supporting information Table S4. GAPDH was used as an internal control. Experiments were done in triplicates, and data analysis was performed using the ΔΔCT method.

Ghamloush F., Ghayad S.E., Rammal G., Fahs A., Ayoub A.J., Merabi Z., Harajly M., Zalzali H, & Saab R. (2019). The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by exosomal miR-486. Scientific Reports, 9, 14242.

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Molecular Mechanisms of Radiation-Induced Cell Death

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To investigate the molecular mechanisms behind high dose radiation, OC explants (n = 6 per condition) were harvested from neonatal rats 6 h after cochlear irradiation (0, 6, 12, and 18 Gy). Total RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) and converted to cDNA using the RT² First Strand Kit (Qiagen). The expression of key cell death genes associated with necrosis and apoptosis were investigated using the Rat Necrosis RT² Profiler PCR Array (#330231; Qiagen) and CFX96 Real-time PCR Detection System (Bio-rad, Hercules, CA, USA), per the manufacturer’s protocol. A list of all the genes and acronyms can be found in Supplemental Table S1. Data was normalized to housekeeping genes and analyzed using the RT² Profiler PCR Array Data Analysis Portal (Version 3.5; Qiagen). The fold change was determined by calculating the ratio between threshold cycle (C_T) of irradiated cochleae and non-irradiated cochleae (0 Gy). Mean fold changes >2-fold were confirmed using real-time polymerase chain reaction (PCR), primer pairs for Bax (R_Bax_1; KiCqStart, Sigma), Fas (R_Fas_1; KiCqStart, Sigma), and Pidd1 (R_Pidd_1; KiCqStart, Sigma), iQ SYBR Green Supermix (Bio-rad) and a CFX96 Real-time PCR Detection System (Bio-rad).

Dinh C.T., Chen S., Nourbakhsh A., Padgett K., Johnson P., Goncalves S., Bracho O., Bas E., Bohorquez J., Monje P.V., Fernandez-Valle C., Elsayyad N., Liu X., Welford S.M, & Telischi F. (2023). Single Fraction and Hypofractionated Radiation Cause Cochlear Damage, Hearing Loss, and Reduced Viability of Merlin-Deficient Schwann Cells. Cancers, 15(10), 2818.

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Quantifying Inflammatory Cytokine Expression in Brain Tissue

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The mRNA expression levels of TNF-α, IL-1β and IL-6 were measured using RT-qPCR. Total RNA was extracted from brain tissues with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The extracted RNA was reverse transcribed into cDNA using the ThermoScript™ RT-PCR system at 40°C for 2 h (Invitrogen; Thermo Fisher Scientific, Inc.). The reaction was performed at 37°C for 30 min, followed by 42°C for 30 min and 85°C for 5 min. Transcripts were quantified by qPCR using a Bio-Rad CFX96 real-time PCR detection system with SYBR-Green IQTM Supermix (both Bio-Rad Laboratories, Inc.). The PCR reaction was performed at 95°C for 3 min, followed by 40 cycles of 95°C for 12 sec and 60°C for 40 sec. An internal control (GAPDH) was used to normalize the expression of the target genes. Quantification of TNF-α, IL-1β and IL-6 levels was conducted using the CFX96™ Real-Time PCR detection system (Bio-Rad Laboratories, Inc.). The primers were purchased from Sangon Biotech Co., Ltd. The following primers were used: TNF-α forward, 5′-ACATACTGACCCACGGCTTC-3′ and reverse, 3′-TCACCCATCCCATCTCTCTC-5′; IL-1β forward, 5′-CAGGCAGGCAGTATCACTCA-3′ and reverse, 3′-AGGCCACAGGTATTTTGTCG-5′; and IL-6 5′-forward, GGCGGATCGGATGTTGTGAT-3′ and reverse, 3′-GGACCCCAGACAATCGGTTG-5′. The relative quantification of the gene expression was calculated using the 2^−ΔΔCq method (20 (link)).

Zhang F.B., Wang J.P., Zhang H.X., Fan G.M, & Cui X. (2019). Effect of β-patchoulene on cerebral ischemia-reperfusion injury and the TLR4/NF-κB signaling pathway. Experimental and Therapeutic Medicine, 17(5), 3335-3342.

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Validating Fungal Transcriptome by qRT-PCR

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To verify the transcriptome data obtained by RNA-Seq, quantitative reverse transcription-PCR (qRT-PCR) was performed in the Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, USA). The fungus A. alternata wild-type Z7 and ΔMetR mutants were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid PDB medium and incubated on a rotary shaker at 25°C and 180 rpm in the dark for 2 days. For RNA extraction, the mycelia of the ΔMetR mutant and wild type were immediately filtered through four layers of cheesecloth, freeze-dried, and ground into a fine powder in liquid nitrogen. Fungal RNA was extracted from each sample using the AxyPrep multisource total RNA miniprep kit (Axygen Biotechnology, Hangzhou, China) according to the manufacturer’s instructions. In total, 5 μg RNA was reverse transcribed into cDNA using the HiScript II Q RT SuperMix kit (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time quantitative PCR was performed using the ChamQ SYBR qPCR master mix kit (Vazyme Biotech Co., Ltd., Nanjing, China). The Bio-Rad CFX96 real-time PCR detection system was used for qRT-PCR with an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s and then annealing and extension at 60°C for 30 s. For each sample, all treatments were performed in triplicate. In this study, the A. alternata actin gene was used as an endogenous control.

Gai Y., Li L., Ma H., Riely B.K., Liu B, & Li H. (2021). Critical Role of MetR/MetB/MetC/MetX in Cysteine and Methionine Metabolism, Fungal Development, and Virulence of Alternaria alternata. Applied and Environmental Microbiology, 87(4), e01911-20.

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