Total RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer’s instructions, then reverse-transcribed using the iScript cDNA synthesis kit (BioRad). Quantitative real-time PCR was performed with an iQreal-time PCR detection system (BioRad) using iQ SYBR Green Supermix (BioRad).
Iq real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The IQ Real-Time PCR Detection System is a laboratory equipment designed for real-time polymerase chain reaction (PCR) analysis. The system provides precise and reliable quantification of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (5 mentions)
Sybr green, by Bio-Rad (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Icycler, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Cybr green, by Bio-Rad (1 mentions)
Rneasy miniprep kit, by Qiagen (1 mentions)
Isolation kits, by STEMCELL (1 mentions)
Random hexamer, by Promega (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
11 protocols using iq real time pcr detection system
1
Total RNA Isolation and qRT-PCR
2
Quantifying Gαq Gene Expression
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Total RNA was extracted from dissected forebrain neocortical tissue using guanidine/cesium chloride ultracentrifugation. First-strand cDNA was synthesized using Murine Moloney Leukemia Virus reverse transcriptase (USB, Cleveland, OH) in conjunction with oligo dT15 primers. Real time PCR was performed using the iQ Real Time PCR detection System (Bio-Rad, Hercules, CA) and SYBR Green (Bio-Rad). Gαq cDNA was amplified using the primers 5′-ACTCTGGAGTCCATCATG3′/TGTATGGGATCTTGAGCG-3′. Cycle thresholds (Ct) of amplification (normalized to β-actin whose Ct values did not differ between groups) were expressed as 1/2ΔCt values so that higher numbers reflect higher expression.
3
Quantifying mRNA Expression in iPSC and Exosomes
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Total RNA was extracted from cultured iPSCs or exosomes using TRIzol Reagent. RNA concentrations and purities were determined using a Nanodrop instrument at wavelengths of 260/280 nm. cDNA was synthesized from 1 μg of total RNA using superscript III (Thermo Fisher Scientific) according to the manufacturer's instructions. The cDNA was diluted with DNase-free water to a concentration of 10 ng/μl. Real time PCR was performed in a Bio-Rad iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using the IQ SYBR Green SuperMix with the following thermal cycles: initial denaturation 95 °C for 5 min, 40 cycles of 20 s at 95 °C, 45 s at 57 °C, and 30 s at 72 °C. Analyses of mRNA levels of target genes were normalized to GAPDH as the internal control and expressed relative to the quantity of the control group. The primers are shown in supplemental table of key resources (Supplementary Table 2 ).
4
Neocortical Gene Expression Analysis
5
Quantitative RT-qPCR Analysis of Brain BDNF
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RNA (10 μg), extracted via guanidine/cesium chloride ultracentrifugation from dissected forebrain neocortex of male and female mice, was reverse transcribed as previously described14 (link). cDNA was amplified using the iQ Real-Time PCR detection system (Bio-Rad, Hercules, CA) and CYBR Green (Bio-Rad) using the Bdnf transcript variants 1 to 3 primer sequences published by Tsankova et al32 (link). Cycle thresholds (Ct) of amplifications (normalized using Ct values obtained for amplification of β-actin) were expressed as 1/2ΔCt.
6
Quantifying Tumor Immune Cell Profiles
7
Quantitative Real-Time PCR Analysis
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Total RNA extraction and cDNA synthesis was carried out as described above. Relative transcripts levels were determined by quantitative real-time PCR (qPCR), using iQ SYBR Green supermix (Bio-Rad), and forward and reverse primers (final concentration: 100nM/qPCR) in a 20 μl final volume (see Table S3 for primer sequences). The qPCR experiments were conducted with the same quantity of embryo equivalent input for all treatments and each sample was run in duplicate using 2 μl of cDNA per reaction. All the samples were analyzed using an iCycler and iQ Real Time PCR Detection System (Bio-Rad). For each standard curve, one reference DNA sample was diluted serially. RNA expression was normalized in relation to the expression of BgActin-5C.
8
Quantitative Analysis of Gene Expression
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Total RNA extraction and cDNA synthesis was carried out as described above. Relative transcripts levels were determined by quantitative real-time PCR (qPCR), using iQ SYBR Green supermix (Bio-Rad), in a 20 μl final volume (see Supplementary Table S2 for primer sequences). The qPCR experiments were conducted with the same quantity of organ equivalent input for all treatments and each sample was run in duplicate using 2 μl of cDNA per reaction. All the samples were analyzed using an iCycler and iQ Real Time PCR Detection System (Bio-Rad). For each standard curve, one reference DNA sample was diluted serially.
9
Quantifying Gene Expression Changes via qPCR
10
Teratoma Formation Assay for iPSCs
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Total RNA was extracted from cultured iPSCs using TRIzol Reagent (Thermo Fisher). cDNA was synthesized from 1 µg of total RNA with SuperScript III (Thermo Fisher), according to the manufacturer's instructions. The cDNA was diluted to 10 ng/μL with DNase-free water. qPCRs were carried out in a Bio-Rad iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using the IQ SYBR Green SuperMix. mRNA levels of target genes were normalized to that of GAPDH as the internal control and were expressed relative to the quantity of the control group.
iPSCs were cultured in complete EC growth medium (ScienCell Research Laboratories, Carlsbad, CA, USA) in the absence of feeder cells and then subcultured once on day 4. On day 6, the cells were mixed with Matrigel and injected subcutaneously (1×106 cells/site) to observe their capability to form teratomas.
iPSCs were cultured in complete EC growth medium (ScienCell Research Laboratories, Carlsbad, CA, USA) in the absence of feeder cells and then subcultured once on day 4. On day 6, the cells were mixed with Matrigel and injected subcutaneously (1×106 cells/site) to observe their capability to form teratomas.
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