Western blot and qPCR were performed as previously described [20 (link)]. The working dilutions of antibodies are listed in Supplementary Table S1 . TRIzol solution (15596-026, Invitrogen, California, USA) and reverse transcription kit (FSQ-101, TOYOBO, Osaka, Japan) were used to examine mRNA level. SYBR™ Select Master Mix (4472908, Applied Biosystems, Foster City, USA) was used for qPCR in 10 μl reaction mixtures in HT 7900 (Applied Biosystems, Foster City, USA). The sequences of primers are listed in Supplementary Table S1 .
Fsq 101
Manufactured by Toyobo
Sourced in Japan, China
The FSQ-101 is a laboratory equipment designed for general use in research and testing environments. It serves as a device for performing various scientific procedures and experiments. The core function of the FSQ-101 is to provide a controlled and consistent platform for conducting analytical and investigative activities.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ht7900, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green real time pcr master mix kit, by Toyobo (1 mentions)
Rna extraction kit, by Magen Biotechnology Co (1 mentions)
Sybr green pcr master mix kit, by Roche (1 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
One step reverse transcription kit, by Takara Bio (1 mentions)
Lightcycle 96 real time pcr system, by Roche (1 mentions)
Abi q6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Rnafast200, by Fastagen (1 mentions)
Pikoreal 96 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Pikoreal software, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Abi 7300 system, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master rox, by Roche (1 mentions)
16 protocols using fsq 101
1
qPCR and Western Blot Analysis
2
Gene Expression Analysis of Muscle Metabolism
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RNA was isolated from GAS samples from the WT and Chrono TG mice. Briefly, 50 mg of GAS tissue was homogenized in 1 mL TRIzol reagent (Life Technologies, Eugene, OR, USA). RNA was carried out following the manufacturer’s instructions. About 1 μg of total RNA was used to synthesize cDNA using a kit (FSQ-101; Toyobo Co., Ltd., Osaka, Japan). The RT-qPCR was performed in an ABI 7500 Real-Time PCR System (Thermo Scientific, Inc., Waltham, MA, USA) using the SYBR Green Real-Time PCR Master Mix kit (Toyobo Co., Ltd., Osaka, Japan). The commercial primers from Qiagen (Hilden, Germany) of Chrono (QT01533749), glucose transporter type 4 (Glut4, QT01044946), glycogen branching enzyme 1 (Gbe1, QT00252924), phosphorylase kinase regulatory subunit alpha 1 (Phka1, QT00143514), hexokinase 2 (Hk2, QT00155582), muscle phosphofructokinase (Pfkm, QT00159754), pyruvate dehydrogenase phosphatase catalytic subunit 1 (Pdp1, QT01165374), TBC1 domain family member 1 (Tbc1d1, QT00156898), pyruvate dehydrogenase kinase 4 (Pdk4, QT00157248), and 18S ribosomal RNA (Rn18s, QT02448075) were used. The mRNA primers of glycogen synthase 1 (Gys1) were synthesized by Thermo Fisher, and the primer sequences are shown in Table 1 . The expression of each sample was calculated using the 2−ΔΔCt method, as described previously [19 (link)].
3
Quantifying Gene Expression with qRT-PCR
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Total RNA from 1×105 cells was first isolated using an RNA extraction kit (R4111-03, Magen) according to the manufacturer’s instructions. cDNA was obtained using a reverse transcription kit (TOYOBO, FSQ-101) and subjected to qRT-PCR. The sequences of primers used were as follows: PRKN-F: 5’-GTGCAGAGACCGTGGAGAAA-3’; PRKN-R: 5’-GCTGCACTGTACCCTGAGTT-3’, CAT-F: 5’-AAAAGATATCATGGCTGACAGCCGGGAT-3’; CAT-R: 5’-AAAAGCGGCCGCTCACAGATTTGCCTTCTC-3’, GAPDH-F: 5’-GACTCATGACCACAGTCCATGC-3’; GAPDH-R: 5’-AGAGGCAGGGATGATGTTCTG-3’. The CT values of GAPDH were used for normalization.
4
Quantifying RNA Expression in Mouse and Cardiomyocytes
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Total RNA samples were isolated from mouse heart or NRVCs using phenol/chloroform and complementary DNA synthesis was performed with high-capacity cDNA reverse transcription reagent kits (FSQ-101, Toyobo, Japan). The SYBR Green PCR Master Mix Kit (04913914001, Roche, Switzerland) was used in quantitative real-time PCR with LightCycler96 Real-Time PCR System (Roche, Switzerland) to quantify target genes. U6 or GAPDH served as an internal control. The primers for quantitative real-time PCR were commercially designed (Table S3 ).
5
TGF-β1 Expression Quantification in Rats
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TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to extract total RNA from rats. cDNA was synthesized from 1 µg of RNA using one-step reverse transcription kit (no. 639505; Takara Bio, Inc., Otsu, Japan). The mRNA levels of each index were measured using a fluorescence quantitative PCR kit (FSQ-101; Toyobo Life Science, Osaka, Japan). GAPDH was taken as an internal reference. TGF-β1 gene localization: NC_000019.10. Primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Sequence: upstream, 5′-GGCCAGATCCTGTCCAAGC-3′ and downstream, 5′-GTGGGTTTCCACCATTAGCAC-3′; internal reference GAPDH: upstream, 5′-TGGCCTTCCGTGTTCCTAC-3′ and downstream, 5′-GAGTTGCTGTTGAAGTCGCA-3′. The relative expression level of each index was calculated by 2−ΔCq [ΔCq = Cq (target gene) - Cq (GAPDH)] (14 (link)–16 (link)).
6
Zebrafish Embryo and P19 Cell RNA Extraction
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Extraction of total RNA from zebrafish embryos of all periods or P19 cells was performed following the standard procedure for TRIzol reagents. Total RNA was extracted from 30 to 40 zebrafish embryos or 1-5 X 107 P19 cells, roughly 1 μg of total RNA was used to reverse transcribe the mRNA to cDNA via a reverse transcription kit (TOYOBO, FSQ-101), and a 2-fold dilution of the cDNA was used for qPCR, with each reaction containing a SYBR Green mix (TOYOBO, QPK-201) performed in a LightCycle 96 Real-Time PCR system (Roche). All reactions with specific forward and reverse primers were performed in triplicate. Primers were listed in Supplementary Table S1 .
7
RNA Extraction and Quantification Protocol
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TRIzol reagent (10296028; Thermo Fisher Scientific) was used to extract the total RNA from the tissues, organoids, and cells. RNA purity and concentration were measured using a spectrophotometer (Nano Drop OneC; Thermo Fisher Scientific). Reverse transcription of RNA to complementary DNA (cDNA) was performed using a cDNA reverse transcription kit (FSQ-101, TOYOBO). Real-time PCR was performed using an ABI Q6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the SYBR Green detection protocol (QPK-201, TOYOBO). Finally, 18 S ribosomal RNA was used as a housekeeping gene, and the 2-ΔΔCT method was used to normalize data. Primers used here are listed (Supplementary Table 1 ).
8
Quantifying Gene Expression via qRT-PCR
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Total mRNA was extracted using RNAfast200 (Fastagen, China) and then converted into cDNA using a reverse transcription kit (TOYOBO, Cat. FSQ-101), according to the manufacturer’s instructions. In order to examine the levels of gene expression, the qRT-PCR analysis provided by the Kit (TAKARA, Cat. RR820A). Internal controls in the form of GAPDH and actin mRNA as housekeeping genes were employed. Table 1
9
RT-qPCR Analysis of Gene Expression
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Total RNA was prepared using the RNeasy Mini kit (Qiagen) and cDNA was synthesized from 0.2–0.5 μg total RNA using a cDNA synthesis kit (FSQ‐101, Toyobo). cDNA was then subjected to RT‐qPCR using pre‐designed gene‐specific primers and probe sets (PrimeTime Std qPCR Assay, IDT) and a reaction mixture (QPS‐101, Toyobo). The primer and probe sequences for the genes are listed in Table S2 . Accumulation of PCR products was monitored in real time by measuring the level of fluorescence (PikoReal 96 Real‐Time PCR System, Thermo Fisher Scientific). Results were analysed by the ΔΔCt method and normalized to GAPDH using PikoReal software (Thermo Fisher Scientific) to determine relative fold changes in gene expression.
10
Quantifying Egr1 Expression in AR42J Cells
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Total RNA from AR42J cell lines was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the isolated RNA was reverse transcribed (cat. no. FSQ-101; Toyobo Life Science): 37°C for 15 min, 50°C for 5 min and 98°C enzyme inactivation reaction for 5 min. RT-qPCR was conducted using FastStart Universal SYBR-Green Master (ROX; Roche Diagnostics) and an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 2 sec and 60°C for 30 sec, with a final extension at 72°C for 10 min. The primers for Egr1 and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd. The relative expression levels of Egr1 were normalized to GAPDH and quantified using the 2−∆∆Cq method (30 (link)). The Egr1 and GAPDH primers were as follows: Egr1 forward, 5′-ACTGGAGGAGATGATGCTGCTGAG-3′ and reverse, 5′-CCGCTGCTGCTGCTGCTG-3′; and GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCAGCCACAGTTC-3′.
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