RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified by absorbance at 260 nm and a standard amount was used as input to an iScript reverse transcriptase reaction (Bio-Rad). The product of this reaction was used for quantitative PCR at a maximum volume of 1 μl per 10 μl final reaction volume. Quantitative PCR was conducted using SsoFast EvaGreen Supermix (Bio-Rad) and run on a CFX384 Real-Time System/C1000 Thermal Cycler (Bio-Rad). Reactions were conducted in triplicate and normalized to β-actin. For each gene, the sample with the highest expression was assigned a value of 1 and other samples were normalized accordingly. Primer sequences are displayed in Table 2 .
Cfx384 real time system c1000 thermal cycler
Manufactured by Bio-Rad
Sourced in United States, Germany
The CFX384 real-time System C1000 Thermal Cycler is a laboratory instrument designed for real-time PCR analysis. It features a 384-well format and a C1000 Thermal Cycler for precise temperature control during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Ssofast evagreen supermix, by Bio-Rad (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Ssofast evagreen, by Bio-Rad (2 mentions)
Hard shell 384 well pcr plates white well clear shell, by Bio-Rad (2 mentions)
Taqman universal master mix with no amperase uracil n glycosylase, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Taqman microrna assays kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna mini kit, by Macherey-Nagel (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Iq5 cycler, by Bio-Rad (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Nucleospin rna isolation kit, by Macherey-Nagel (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using cfx384 real time system c1000 thermal cycler
1
Quantitative gene expression analysis
2
Quantitative Real-Time PCR Analysis of CPEB4 Isoforms
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Quantification was performed by real-time PCR using a CFX 384 Real Time System C1000 Thermal Cycler (Bio-Rad) in combination with SsoFast Eva Green (Bio-Rad, CN 172-5204) and 0.25 μM of primer pair was used. Data were analyzed by GenEx 5.3.7 software (Multid AnaLyses AB). The mRNA levels were normalized first relative to total RNA and then relative to the 18S ribosome subunit, β-ACTIN, GAPDH and β-TUBULIN gene expression in each sample. Absolute quantitative PCR was performed to determine the percentage of each CPEB4 splicing isoform in both human and mouse species using specific primers (Supplementary Table 7 ). For every primer couple, specificity was tested, PCR assay conditions were adjusted to obtain a single amplicon analyzed by both melting curve analysis and agarose gel electrophoresis. Amplicons of each CPEB4 isoform were serially diluted to generate a calibration curve. A duplication of this curve was made to give robustness. Next, total tissue RNA was extracted, and quantitative real-time RT-PCR was performed. Finally, the percentage of each CPEB4 isoform with respect to total CPEB4 copies was calculated.
3
Quantitative Analysis of Tumor-Associated MicroRNAs
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Using the instructions of TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, USA), 10 ng of the total RNA were reverse transcribed. Small nucleus RNA RNU6B, hsa-miR-125b, hsa-miR-425-5p, hsa-miR-21, hsa-miR-200c, hsa-miR-183, hsa-miR-182 and hsa-miR-100 probes and primers were ordered as part of the TaqMan microRNA Assays Kit (Applied Biosystems, USA). cDNA synthesis was performed in a multiplex reaction in which two miRNA primers were used along with the endogenous control (RNU6B). RT-qPCR was performed by means of BioRad CFX384 Real Time System, C1000 Thermal Cycler (Germany). Each well consisted of 5 μL of 2x TaqMan Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, USA), 2 uL of RNase free water, 0.5 μL of corresponding 20x miRNA probe and 2.5 μL of the cDNA. The reactions were completed in duplicates for each microRNA probe.
Tumor tissues were normalized according to the normal adjacent tissues in the same RT-qPCR run to ensure inter-run calibration. The conditions of cycling were 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and an annealing temperature of 60°C for 60 seconds.
By means of the ΔΔCt equation, the expression of experimental miRNA in tumor tissues was calculated in comparison to the normal adjacent tissue (NAT) samples using the endogenous control RNU6B.
Tumor tissues were normalized according to the normal adjacent tissues in the same RT-qPCR run to ensure inter-run calibration. The conditions of cycling were 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and an annealing temperature of 60°C for 60 seconds.
By means of the ΔΔCt equation, the expression of experimental miRNA in tumor tissues was calculated in comparison to the normal adjacent tissue (NAT) samples using the endogenous control RNU6B.
4
Adrenal Gland RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from frozen adrenal glands with the TRI Reagent (MRC) after mechanical tissue disruption, extracted with chloroform and the NucleoSpin RNA Mini kit (Macherey-Nagel). Total RNA from sorted cells was isolated with the Rneasy Plus Micro Kit (QIAGEN) according to the manufacturer’s instructions. cDNA was synthesized with the iScript cDNA Synthesis kit (Bio-Rad) and gene expression was determined using the SsoFast Eva Green Supermix (Bio-Rad), with a CFX384 real-time System C1000 Thermal Cycler (Bio-Rad) and the Bio-Rad CFX Manager 3.1 software. The relative gene expression was calculated using the ΔΔCt method, 18S was used as a reference gene. Primers are listed in Table 5 .
5
Real-time PCR Protocol for Gene Expression
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Total RNA was extracted following the protocol described previously [96 (link)]. RNA samples were treated with RQ1 RNase-Free DNase (Promega), and cDNA synthesis was performed with iScript Reverse Transcription Supermix (Bio-Rad) in the presence of oligo (dT) or random primers. Real-time PCR was performed in a reaction volume of 10 μl containing cDNA (10 ng) and 5 μl of SsoFast EvaGreen Supermix (Bio-Rad) using a CFX384 Real- Time System C1000 Thermal Cycler (Bio-Rad) with annealing at 60 °C for 10 s and extension at 72 °C for 15 s. The reactions were run for 40 cycles, and after the final cycle, a melting curve was performed to verify the reaction specificity. Actin, elongation factor-1-alpha (EF1-α), and 40S ribosomal protein were used as reference genes in pine, and actin was used as a reference gene in N. benthamiana samples. The fluorescence of the PCR products was continuously monitored using an iQ5 cycler (Bio-Rad), and relative gene expression was estimated as previously described [94 (link)]. The gene-specific primers used are described in Additional file 4 : Table S1.
6
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted from cells or tissues using the Nucleospin RNA isolation kit (Macherey-Nagel, Dueren, Germany), according to the manufacturer’s instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qPCR was performed using the SsoFast Eva Green Supermix (Bio-Rad Hercules, CA, USA), a CFX384 real-time System C1000 Thermal Cycler (Bio-Rad), and the Bio-Rad CFX Manager 3.1 software, as previously described [36 (link),38 (link),43 (link)]. The relative amount of mRNA was calculated with the ΔΔCt method, using 18s as a housekeeping gene. The primer sequences are listed in Table S1 .
7
Fluorescent Probe-Based AP Lyase Assay
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The assay was performed using as substrate 100 nM of the indicated 2′-deoxyuridine-containing fluorescently labeled probe, that was treated with E. coli UDG (2 U) for 60 min at 37°C in 30 mM HEPES (pH 7.5) and 4% glycerol to obtain natural AP sites. Afterwards, the mixture was supplemented with 5 mM MnCl2 and 10 μM of GTP. The AP lyase activity was started with the addition of 400 nM of the wild-type BsuLigD, in a final volume of 20 μl. During the incubation at 30°C, fluorescence intensity was acquired once per cycle (20 s per cycle) in a CFX384 Real Time System C1000 Thermal Cycler (Bio-Rad), in hard-Shell® 384-Well PCR Plates White Well Clear shell (Bio-Rad CN HSP-3805).
8
Quantifying miRNA Expression via RT-qPCR
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To detect miRNA expression, RT-qPCR (BioRad CFX384 Real Time System, C1000 thermal cycler, Hercules, CA, USA) was carried out in duplicate for each sample using 2x TaqMan® Universal Master Mix with no Amperase Uracil N-Glycosylase (UNG) (Applied Biosystems, Waltham, MA, USA) including a ‘no template’ control for each miRNA, following manufacturer protocols. The cycling conditions were 10 min at 95 °C, then, 40 cycles of 15 s at 95 °C (denaturing step) and 60 s at 60 °C (annealing and extension steps). The miR-16 showed the same expression in plasma from tumor and healthy samples with a coefficient of variation (CV) = 8.7% and was used as an endogenous control. Similarly, miRNA molecules of interest showed constant expression in all healthy subjects. Therefore, the relative expression of selected miRNA was normalized to the chosen endogenous control and was compared to healthy controls. The relative fold change of expression was calculated using the ∆∆Ct equation.
9
Quantitative Analysis of Chemokines
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RNA was isolated from the cells with an RNeasy kit (Qiagen) according to manufacturer’s protocol. Isolated RNA was quantified with Nanodrop (Thermo Scientific). Transformation of RNA into cDNA was performed with a high capacity RNA-to-cDNA kit (Applied Biosystems). cDNA (5–10 ng per well) was analyzed by SYBR green real-time PCR with 10 nM primers using a CFX96 or CFX384 Real-Time System C1000 Thermal Cycler (Bio-Rad). The primers used for qPCR are shown in the table below. To measure the chemokine and chemokine receptors, mouse chemokines and receptors RT2 Profiler PCR Array Mouse Chemokines and receptors was purchased from Qiagen (online supplementary file 2 ).
10
RNA Isolation and Quantitative PCR
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RNA was isolated from frozen tissues with the TRI Reagent (Molecular Research Center, Inc.) after mechanical tissue disruption or from cells with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA obtained with TRI Reagent was subsequently extracted with Chloroform and the NucleoSpin RNA, Maxi kit (Macherey-Nagel). Reverse transcription was performed with the iScript cDNA Synthesis kit (Bio-Rad), and cDNA was analyzed by qPCR using the SsoFast Eva Green Supermix (Bio-Rad), a CFX384 real-time System C1000 Thermal Cycler (Bio-Rad) and the Bio-Rad CFX Manager 3.1 software (61 (link)). Primers used are listed in table S6.
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