In order to confirm the gene expression results obtained from the microarray, the expression level of 4 randomly selected differentially expressed lncRNA (Gm11985 and Ak156531) and mRNAs (Igfbpl and Npas4) were further analyzed using RT-qPCR assay as shown in our previous publication [19 (link)]. Briefly, cDNA was synthesized using an RT2 First Strand Kit (Qiagen). For qPCR assay, cDNA was mixed with ™ SYBR Green Master Mix (Thermo Fisher Scientific), primers, and pure water (Qiagen). Triplicate samples were loaded into 96-well plates (25 μl/well] and RT-qPCR was performed using QuantStudio™ 6 Real-Time PCR detection system (Thermo Fisher Scientific] for 10 minutes at 95°C followed by 40 cycles of a denaturation step (15 seconds at 95°C] and a combined annealing/extension step (30 seconds at 60°C]. The mean cycle threshold (Ct) values of triplicate wells for each sample were collected and the expression data was normalized to the endogenous control (beta-actin). Melting curves were monitored to validate the purity of the PCR product in each well.
Quantstudio 6 real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 6 Real-Time PCR detection system is a real-time PCR instrument designed for accurate and reliable quantification of nucleic acid samples. It utilizes fluorescence-based detection technology to monitor the amplification of target DNA sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Pure water, by Qiagen (1 mentions)
Sybr green realtime pcr master mix plus, by Vazyme (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Primer 5, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast qpcr master mix 2x kit, by Roche (1 mentions)
5 protocols using quantstudio 6 real time pcr detection system
1
Validation of Microarray Gene Expression
2
Quantification of RUNX2 and RANKL Gene Expression
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Total RNA was extracted from rat caput femoris by using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA concentrations were determined by the absorbance ratio of 260/280 nm. Then, 1 µg of the extracted RNA was reverse-transcribed into first-strand complementary DNA (cDNA) using a ReverTra Ace® qPCR RT kit (cat. no. R101-01/02; Vazyme Biotech Co., Ltd.). Gene expression of RUNX2 and RANKL were quantified by using a SYBR-Green Real time PCR Master Mix Plus (Q111-02; Vazyme Biotech Co., Ltd.) following the manufacturer's protocols. Measurements were conducted in triplicate under standard reaction conditions and normalized to β-actin. Primers were obtained from TsingKe Biological Technology Co., Ltd. All temperature cycling (two initial denaturation steps at 50°C for 2 min, then 95°C for 10 min, and followed by 40 cycles at 95°C 30 sec and 60°C 30 sec for amplification) and gene amplification were conducted in a QuantStudio™ 6 Real-Time PCR Detection System (Thermo Fisher Scientific, Inc.). The primer sequences used are presented in Table I .
3
RNA Extraction and qPCR Analysis
4
Quantitative Analysis of Cerebral Organoid Development
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A total of 5 ng cDNA, Power up SYBR Green Master Mix (Thermo Fisher Scientific), primers, and water (Qiagen) were loaded in triplicate in 384-well plates (10 μL/well) and PCR was performed using QuantStudio™ 6 Real-Time PCR detection system (Thermo Fisher Scientific). Primers were designed for MAP2, OCT4, S100B, SCN1A, SCN2A, SCN3A, SCN8A, SCN9A (SCN gene family members encode for respective Nav 1 family of sodium channels) [20 (link),21 (link)], and SMA, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene (Table 1 ). The cycling conditions were 10 min at 95 °C, 40 cycles of denaturation (15 s at 95 °C) and a combined annealing/extension step (30 s at 60 °C). The mean cycle threshold (Ct) values of triplicates within each sample were detected and the expression data were normalized to the GAPDH housekeeping gene. Melting curves were monitored to validate the purity of the PCR product in each well. The 1-month and 2-month-old cerebral organoid gene abundance is expressed as a% change from iPSCs.
5
Quantifying Aortic Valve Gene Expression
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Specimens from aortic valves were cut into pieces and smashed with liquid nitrogen. Total RNAs from leaflets were prepared using Trizol reagent (Thermo Fisher, Waltham, MA, USA). The supernatants containing total RNAs were then purified using Direct-zol RNA MicroPrep Kit (Zymo Research). The concentration of total RNAs was measured using NanoDrop (Thermo Fisher, Waltham, MA, USA) and 0.1–1 µg of total RNAs were reverse transcribed with cDNA Synthesis Kit (Thermo Fisher, Waltham, MA, USA) in accordance with the manufacturers’ instruction. The cDNAs were further sixfold diluted and subjected into RT-qPCR experiments using QuantStudio 6 Real-Time PCR Detection System (Thermo Fisher; Waltham, MA, USA) and KAPA SYBR FAST qPCR Master Mix (2X) Kit (Kapa Biosystems, Wilmington, DE, USA). Primers for RT-qPCR were designed using Primer 5.0 software and synthesized by Invitrogen. Expression was normalized to GAPDH. The data were analyzed using the 2-ΔΔCT method.
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