After obtaining brain tissue from mice at different ICH time points (n=6), total RNA was extracted using a total RNA extraction kit (DP431, TIANGEN), and the concentration and purity were determined using a NanoVue Plus Micro-Volume UV-Vis spectrophotometer. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit (FSQ-201, TOYOBO), and the concentration and purity of the cDNA were determined. Finally, 20μL of amplification reaction mix (QPS-201, TOYOBO) was prepared and the samples were introduced into an ABI StepOne Real-Time PCR system. The primer sequences are listed in Table 2 . The ΔΔCT method was used to calculate the results. The data obtained were analyzed by one-way ANOVA. Tukey multiple comparison was used to test for statistical significance between groups.
Fsq 201
Manufactured by Toyobo
Sourced in Japan
The FSQ-201 is a laboratory equipment product manufactured by Toyobo. It serves as a tool for scientific analysis and experimentation within research and development settings. The core function of the FSQ-201 is to provide precise measurement and data collection capabilities for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Dp431, by Tiangen Biotech (1 mentions)
Qps 201, by Toyobo (1 mentions)
Lightcycler, by Roche (1 mentions)
Golden star t6 super pcr mix, by Tsingke (1 mentions)
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (1 mentions)
Dp424, by Tiangen Biotech (1 mentions)
Real time pcr kit, by Toyobo (1 mentions)
Steponeplus rt pcr system, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Flx800t, by Agilent Technologies (1 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Lightcycler 1.5 real time pcr system, by Roche (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Toyobo (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
10 protocols using fsq 201
1
Quantitative RT-PCR Analysis of ICH
2
Quantitative RNA Expression Analysis
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Total RNA was prepared using the TRIzol reagent (cat. no. 15596018; Invitrogen), according to the manufacturer’s instructions. Total RNA was extracted from animal tissues or cell lines, and cDNA was prepared using a reverse transcription kit (FSQ-201; TOYOBO). The primers used for each gene are listed in Table S3 . For RT-PCR, 25–30 cycles of PCR were performed with Golden Star T6 Super PCR Mix (Tsingke). Quantitative real-time PCR (qRT-PCR) was performed in a Roche LightCycler and the ΔΔCt method was used to analyze the data.
3
Macrophage Polarization and Stem Cell Oxidative Stress
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RAW 264.7 was seeded in a 24-well plate at 2 × 105 cells per well and cultured with scaffold-conditioned medium for 24 hours. Then, LPS (100 ng/ml; BS904, Biosharp, Anhui, China) was added to the medium for 24 hours to induce M1 macrophage polarization. MSCs were seeded in a 12-well plate at 5 × 104 cells per well and cultured with scaffold-conditioned medium for 24 hours. Then, 200 μM H2O2 was added to the medium for 24 hours to mimic the pathological microenvironment. TSPCs and C2C12 were seeded in a 12-well plate at 1 × 104 cells per well and cultured with scaffold-conditioned medium for 24 hours. Then, IL-1β (10 ng/ml) was added to the medium for 7 days to mimic the pathological microenvironment. The groups without LPS/H2O2/IL-1β stimulation were defined as the control group (Ctrl). The total RNA was extracted, and the gene expression was analyzed using a real-time qPCR assay. The reagents used were listed as follows: total RNA extraction kit (DP424, Tiangen, Beijing, China), cDNA reverse transcription kit (FSQ-201, Toyobo, Japan), and qPCR kit [SYBR Green Premix Pro Taq HS qPCR Kit, AG11718, Accurate Biotechnology (Hunan) Co., Ltd., China]. All of the procedures were performed following the manufacturer’s protocol. Primer (Genscript, Nanjing, China) sequences used in this study are summarized in Table 1 .
4
Quantitative Analysis of GmWAK1 in Soybean
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We performed quantitative real-time PCR (qRT-PCR) to analyze the GmWAK1 transcript levels using a real-time PCR kit (Toyobo, Japan; part number FSQ-201) [83 (link)]. As a housekeeping gene, we used GmEF1β (GenBank accession no. NM_001248778) in soybean as an internal reference to normalize all data. We calculated relative transcript levels using the 2−ΔΔCT method. We performed three biological replicates per experiment per line.
5
Quantifying Cell Lineage Markers
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The expression levels of connexin 43 (CX43), smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin (SMTN) were analyzed by RT-qPCR. Briefly, total RNA was isolated using TRI reagent (Sigma-Aldrich), according to the manufacturer's instructions. The purity and concentration of the isolated RNA were determined using a spectrophotometer (FLX800T; Biotek, VT, USA). cDNA synthesis was performed using 500 ng RNase-free, DNase-treated, total RNA using a reverse transcription system (FSQ-201; Toyobo, Osaka, Japan). Gene expression levels were measured by the comparative Ct method using the StepOne Plus RT-PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH expression was used as an internal control. The primers used in this study are listed in Table S2 .
6
RNA Extraction and RT-qPCR Analysis
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Trizol Reagent (Invitrogen, 15596026) was used to extract total RNAs. 2μg total RNAs were reversely transcribed into cDNAs using a reverse transcription kit (TOYOBO, FSQ-201). Then the cDNAs could then be used as a template for RT-qPCR utilizing the SYBR-Green Master PCR Mix (TOYOBO, QPK-201) and unique primers on a 7500 Fast Real-Time PCR System (Applied Biosystems). Hprt served as the internal control. Table 1 contains the primers ultilized.
7
Quantification of Gene Expression
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To quantify gene expression, each cultured cell line was harvested using the Trizol reagent (Qiagen 79306) in accordance with the manufacturer's instructions. Total RNA was purified with chloroform and precipitated with isopropanol. Purified RNA was resuspended in DEPC water. RNA (600 ng) was incubated with 10 pmol random hexamer and a High Capacity cDNA reverse transcription kit (Toyobo FSQ-201) to achieve reverse transcription. RT-PCR was performed using primers listed in Supplementary Table S1 , according to the protocol previously described (29 (link)). The expression ratio was calculated using the image J program with Gapdh as the internal reference gene. Real-time quantitative PCR (qPCR) was done using SYBR green (TaKaRa RR420A) and Light cycler 1.5 real-time PCR system (Roche). The relative mRNA expression levels were determined with the 2-ΔΔC(t) method. Errors were calculated from at least two independent experiments. Primer sequences for RT-qPCR used are listed in Supplementary Table S1 .
8
Validating RNA-Seq Data by qRT-PCR
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qRT-PCR was employed to validate the RNA-Seq data. Total RNA of 500 ng was reverse-transcribed into cDNA using a first-strand cDNA synthesis kit (Toyobo, Tokyo, Japan; FSQ-201). The expression levels of the eight hub genes were quantified by qRT-PCR using a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) and a SYBR green master mix (Takara, Mountain View, CA, USA). The relative expression levels of target genes were determined by the 2−ΔΔCt method. The housekeeping gene MsACTIN was used as the internal control. Primers for specific genes are presented in Table S1
9
RNA Isolation and Reverse Transcription
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Twenty spheroids from each condition were lysed using 250 µL of Trizol reagent (T9424, Sigma-Aldrich) in a microfuge tube and incubated for 5 min at room temperature. Fifty µL of chloroform (033-15721, FUJIFILM Wako) was then added to the sample, vortexed, and incubated for 10 min at room temperature. The microfuge tubes were centrifuged at 4 °C for 15 min at 12,000×g and 100 µL of each aqueous phase was transferred to a new microfuge tube followed by the addition of 125 µL of 2-propanol (162-17001, FUJIFILM Wako), vortexing, and incubation for 5 min at room temperature. The tubes were centrifuged at 12,000× g at 4 °C for 10 min, the supernatant removed, and the RNA pellet at the bottom of each tube washed with 75% ethanol (052-07221, FUJIFILM Wako). After the removal of the ethanol following centrifugation at 7500× g at 4 °C for 5 min, the pellet was dried and then dissolved in 30 µL of RNase-free water. RNA was quantified using a Nano Drop (ND-1000, Thermo Fisher Scientific) and then denatured by incubation at 65 °C for 5 min before being placed on ice. Reverse-transcription was performed using 8 µL RNA solution containing 500 ng of RNA and 2 µL of reagents and primers (FSQ-201, TOYOBO CO., LTD., Osaka, Japan) following the manufacturer’s instructions.
10
Quantitative Real-Time PCR Analysis
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Total RNAs were extracted from the cells with TRIzol (Invitrogen). The amount of RNA was quantified spectrophotometrically with a Nano-Drop ND-1000. Reverse transcription of total RNAs into cDNA was performed with a reverse transcriptase kit (FSQ-201, TOYOBO, Japan). The relative mRNA expression was determined by real time PCR using THUNDERBIRD SYBR qPCR Mix (QPS-201, TOYOBO, Japan). Briefly, a 20 µL qPCR system was performed for 40 cycles according to the following conditions: An initial denaturation was performed at 95 • C for 3 min, followed by denaturation at 95 • C for 15 sec, annealing at 58 • C for 30 sec, and extension at 72 • C for 7 min. Relative quantification of mRNA expression was calculated us-ing the 2 -∆∆Cq method. 18S was used as a housekeeping gene. The mRNA primer sequences were list in Supplementary Table 3.
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