The RNA was transcribed to cDNA using the Script cDNA synthesis kit (Cat. No. PCR-511L, Jena Bioscience) according to the manufacturer’s instructions. In brief, around 1000 ng of RNA was added to the cDNA synthesis reaction mix before incubation using the thermal cycler at 50°C for 60 min. The resulting cDNA was stored at –20°C. The cDNA was used in a real-time PCR reaction using a real-time green master mix (SYPER Green equivalent) with a lowROX kit (Cat. No. PCR-316L, Jena Bioscience), according to the manufacturer’s instructions. To give a brief summary of the process, about 1000 ng of cDNA and the primer set were added to the PCR reaction mix. After brief centrifugation, the reaction was run using the qTOWER³ real-time PCR system (Analytik Jena, Jena, Germany) available at Stem Cells Research Center at College of Medicine at King Saud University. The thermal conditions were 10 min at 94°C for initial denaturation and polymerase activation, followed by 40 cycles of 20 s denaturing (94°C) and 60 s annealing/elongation (62°C). The primer sequences are listed in Table I . All reactions were run in triplicate. The β-actin mRNA expression level was used to normalize the mRNA expression of the other genes. The comparative Ct (2–∆∆Ct) method was used to calculate the fold change in the expression of the genes of interest.
Qtower real time pcr system
Manufactured by Analytik Jena
Sourced in Germany
The QTOWER real-time PCR system is a laboratory instrument designed for the amplification and detection of nucleic acids. It provides precise and reliable real-time PCR (qPCR) analysis capabilities. The core function of the QTOWER is to perform quantitative polymerase chain reaction (qPCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Taqman real time pcr assay, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Hot firepol evagreen qpcr mix plus, by Solis BioDyne (1 mentions)
Firescript rt cdna synthesis mix, by Solis BioDyne (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dnase, by Qiagen (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Trizon reagent, by CWBIO (1 mentions)
Sybr green master mix, by Novogene (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Hiefftm qpcr sybr green master mix, by Yeasen (1 mentions)
Primerscripttm rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Transstart green qpcr supermix kit, by Transgene (1 mentions)
15 protocols using qtower real time pcr system
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Mouse Hippocampus Gene Expression Analysis
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After identifying social ranks, mice were sacrificed for isolation of the hippocampus. Total RNA was extracted using RNeasy Mini Kit (Qiagen), including DNase (Qiagen) treatment. cDNA of mRNA was generated by FIREScript® RT cDNA Synthesis Mix (Solis Biodyne) using oligo dT as the primer. The qPCR reactions were performed under a qTOWER³ real-time PCR system (Analytik Jena) using 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis Biodyne) following the manufacturer’s protocol. The relative expression was calculated using the ΔCT method, and GAPDH was used as a normalization control. The primer sequences used for qPCR are shown in Table S5.
3
Silencing mmp13 in Diseased Chondrocytes
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Three siM13 sequences were synthesized to transfect diseased chondrocytes and mmp13 RNA expression was measured via RT-qPCR assay. Briefly, diseased chondrocytes were induced, as mentioned above. The siRNAs were mixed with Lipofectamine 3000 in DMEM/F12 complete medium and added to diseased chondrocytes for 24 h of incubation at a final siM13 concentration of 5 μg/mL. Subsequently, the total RNA was extracted using Trizon reagent (Cwbio, China), and the cDNA was obtained through a reverse transcription kit (Takara#RR036A, Takara Bio USA). The RT-qPCR assay was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820, Takara Bio, USA) at the qTOWER³ real-time PCR system (Analytik Jena, Jena, Germany). GAPDH served as an internal reference. The relative expression of mmp13 mRNA was calculated using the 2−ΔΔCt methodology.
4
Quantitative Analysis of Nrf2 Pathway
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PCR reactions were performed with a qTower real‐time PCR System (Analytik Jena, Jena, Germany). Relative quantification was performed by the ΔΔCT method, normalizing target gene expression on GAPDH as housekeeping gene. The following TaqMan® real‐time PCR assays from Thermo Fisher Scientific were used: KEAP1 Hs00202227_m1, NFE2L2 Hs00975961_g1, NQO1 Hs02512143_s1, AKR1C1 Hs04230636_sH, HMOX1 Hs01110250_m1, HCAR2 Hs02341584_s1, GAPDH Hs02758991_g1.
5
Quantitative Gene Expression Analysis in Cucurbitaceae
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The RNA Simple Total RNA Kit (Tiangen, China) and ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) were used for total RNA extraction and cDNA synthesis, respectively. Speci c primers for candidate genes (Cla97C01G008760 and 196920), transcription factors (TFs; Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130) and carotenoid metabolic pathway genes [ClZDS (Cla97C06G118930), ClNCED-7 (Cla97C07G137260), ClCRTISO (Cla97C10G200950), ClCHYB (Cla97C05G090480), ClLCYB (Cla97C04G070940), ClPDS (Cla97C07G142100), ClNCED-1 (Cla97C01G024630), and ClCHXE (Cla97C01G002480)] were designed for gene expression analysis through quantitative real-time polymerase chain reaction (qRT-PCR) (Table 3). SYBR Green Master Mix (Novogene, Beijing) was used to perform the qRT-PCRs in the QTOWER Real-Time PCR System (Analytik Jena, Germany) according to the manufacturer's instructions. qRT-PCR ampli cation and mixing were performed as previously described. Each experiment was performed with three biological repetitions and three technical repetitions, and relative gene expression levels were determined using the 2 ΔΔCT method (Bustin et al 2009).
6
Tracheal Total RNA Extraction and qRT-PCR
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Total RNA of trachea was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) and the corresponding kit (Omega, Norcross, GA, USA). Reverse transcription was performed by using Reverse Transcriptase M-MLV (Takara, Kusatsu, Japan). Quantitative real-time PCR (qRT-PCR) was performed using the HieffTM qPCR SYBR® Green Master Mix (Yeasen, Shanghai, China) and qTOWER real-time PCR system (Analytik Jena, Jena, FT, Germany). The reaction solution of qRT-PCR was 10 μL, contained 5 μL of SYBR Green, 0.8 μL of first strand cDNA template, 0.3 mM of each primer, and 5 μL ddH2O. The eukaryotic translation initiation factor 4A was used as the internal control, which microarray probe ID is sw22934 [37 (link)]. The primers were listed in Supplementary Table S1 . Three biological replicates were performed. The secondary branch of the trachea from at least three individuals were obtained and washed three times with 1× PBS in ice bath and observed under a microscope to ensure that other tissues were cleaned.
7
Plant RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from treated leaves using a plant RNA kit (boer, ChongQing, China) according to the manufacturer’s instruction. First strand cDNA was synthesized from 1 µg of total RNA using the PrimerScriptTM RT reagent kit with gDNA Eraser (Takara, Japan). The cDNA samples were diluted five times in sterile water before storing at −20 °C. The program for the qRT-PCR was as follows: 5 min at 95 °C, 40 cycles of 5 s at 95 °C and 30 s at 62 °C, 30 s at 95 °C. qRT-PCR was performed in a total volume of 20 μL (0.4 μL each primer (10 μM), 4 μL cDNA, 10 μL SYBR Green Master mix (2×), and 5.2 μL ddH2O) in three technological replicates on a QTOWER Real Time PCR System (Analytik Jena, Germany), and the relative expression of the genes was calculated using the 2−ΔCt method [41 (link)]. GhACTIN was used as the internal standard.
8
Astrocyte Gene Expression Quantification
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Gene expression was analysed by real time PCR. Cultured astrocytes were lysed in RLT-buffer (RNeasy kit, QIAGEN, Hilden, Germany) and total RNA was isolated using the RNeasy kit following the manufacturer’s instructions (QIAGEN). RNA yield was quantified by absorbance measurements at 260 nm. 50–500 ng of total RNA were used per reaction to reversely transcribe RNA into cDNA, using QuantiTect transcriptase according to the protocols (QIAGEN). PCR reactions were performed at a 5 µl scale on a qTower real time PCR System (Analytic Jena) in triplicates. Relative quantification was performed by the ΔΔCT method, normalizing target gene expression either on Actb/β-Actin or Rn18s as housekeeping genes. Custom made primers were used to amplify murine slc1a3 mRNA (Thermo Fisher Cat. # 4331182) or murine slc1a2 mRNA (Thermo Fisher Cat # 4331182).
9
Quantitative Analysis of Synergistic Effect
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To explore the molecular mechanism underlying the BBH and FLC synergistic effect, quantitative reverse transcription PCR (qRT-PCR) experiments were performed (Haque et al., 2016 ). C. albicans cells were cultured in YPD medium and diluted to 1 × 105 CFU/mL with RPMI 1640 medium. Cells were incubated overnight with agitation (200 rpm) at 35°C with 2 μg/mL BBH and 1 μg/mL FLC. Then cells were washed and harvested for RNA extraction. Total RNA was isolated using a TRIzol RNA isolation kit (Invitrogen, United States). cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (Transgen, China) for qPCR. Target gene and endogenous control (actin1) primers were designed and synthesized by Shanghai Biotech (Supplementary Table S1 ). The qRT-PCR reaction system was mixed with cDNA, gene primers, and TransStart Green qPCR SuperMix kit (Transgen, China) in 20 μL reaction. qRT-PCR was carried on a qTower real-time PCR system (Analytik Jena, Germany) with an initial denaturation at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, annealing at 59°C for 15 s, and extension 72°C for 10 s. Primer specificity and optimal annealing temperature were determined using melt-curve analysis. Relative target gene expression fold changes were calculated by the 2–ΔΔct method.
10
Quantifying Rice ABA Biosynthetic Genes
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Total RNA was isolated from wild-type (cv. Nipponbare) primary root tips (1 cm, encompassing the meristematic and elongation zones) of 7-d-old plants using TRIzol reagent (Applied Biosystems) as per the manufacturer’s protocol. To remove genomic DNA contamination, isolated RNA was treated with DNase1 enzyme (1 U µg−1 of total RNA). One microgram of total RNA from each of three independent biological replicates (each replicate comprised 10 to 15 root tips) was used for cDNA synthesis using RevertAid First-strand cDNA synthesis kit (Thermo Fisher) according to the manufacturer’s instructions.
SensiMix SYBR Hi-ROX (Bioline) master mix was used for qRT-PCR on a qTOWER real- time PCR System (Analytik Jena GmbH) with three biological replicates per treatment. Expression values were normalized with expression values of an endogenous control, ubiquitin5 (Os01g0328400). Relative expression levels (fold change; FC) were calculated using the 2-Δ(ΔCt) method with respect to the control, as described previously (35 (link)). Primers used for rice ABA biosynthetic genes and their expression analysis are listed inSI Appendix, Table S1 . All FCs were statistically evaluated using Student’s t test.
SensiMix SYBR Hi-ROX (Bioline) master mix was used for qRT-PCR on a qTOWER real- time PCR System (Analytik Jena GmbH) with three biological replicates per treatment. Expression values were normalized with expression values of an endogenous control, ubiquitin5 (Os01g0328400). Relative expression levels (fold change; FC) were calculated using the 2-Δ(ΔCt) method with respect to the control, as described previously (35 (link)). Primers used for rice ABA biosynthetic genes and their expression analysis are listed in
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