Cells were incubated with the selected alkaloids for 24 h prior to RNA extraction. Total RNA was extracted from HeLa cells with the FavorPrep™ Total RNA purification mini kit (Favorgen, Ping Tung, Taiwan). cDNAs were synthesised by performing reverse transcription with SuperScript® VILO™ Master Mix (Invitrogen, Grand Island, NY, USA). Real-time PCR was carried out on a ViiA™ 7 Real Time PCR System (Applied Biosystems, Grand Island, NY, USA) using the FS Universal SYBR Green Master Mix (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. PCR was carried out with the p62 primers 5′-GGA GCA GAT GAG GAA GAT CG-3′ and 5′-GAC GGG TCC ACT TCT TTT GA -3′.
Fs universal sybr green master mix
Manufactured by Roche
Sourced in United States
The FS Universal SYBR Green Master Mix is a ready-to-use solution for real-time PCR applications. It contains all the necessary components for the detection and quantification of DNA targets using SYBR Green chemistry.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Favorprep total rna purification mini kit, by Favorgen Biotech (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Rat igg1, by BioLegend (1 mentions)
Anti mouse il 6 antibody, by BioLegend (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
5 protocols using fs universal sybr green master mix
1
HeLa Cell p62 Gene Expression
2
Quantifying RNA and Protein Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen), and reverse transcription was performed using PrimeScript reverse transcriptase (TaKaRa, Japan). qRT-PCR was conducted with FS Universal SYBR green master mix (Roche) on a CFX96 Touch real-time PCR detection system (Bio-Rad, USA), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control for the purpose of normalization. For Western blotting, cells were washed three times with phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer (1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride [PMSF] in PBS) on ice. After being separated by SDS-PAGE, the proteins in the lysates were electrotransferred to polyvinylidene difluoride (PVDF) membranes and then immunoblotted with the indicated antibodies. GAPDH was used as a loading control.
3
Quantifying Tobacco Gene Expression by Real-Time RT-PCR
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First-strand cDNA synthesis was performed with 4 μg of total RNA using the Revert Aid First Strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The transcriptional profiles of selected genes were analyzed via real-time RT-PCR using the FS Universal SYBR Green master mix (Roche) and a 7500 real-time PCR system (Applied biosystems). The PCR involved 95 °C hold for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 35 s. The relative expression ratio values for different development time points relative to the first sampling time point (15DAT) were calculated using 2−ΔΔCT method. The Actin gene of N. tabacum was used as a reference gene for normalization of the expression. The primers G0571 (5′-ACCTCTATGGCAACATTGTGCTCAG-3′) and G0572 (5′-CTGGGAGCCAAAGCGGTGATT-3′) were used for Actin7 gene qRT-PCR assay. The primers G0585 (5′-CAGTGGCTAATCAACCTGTTTCGG-3′) and G0586 (5′-ACACCACTTGAATAGAACTGGAAATCG-3′) were used for CP1 gene qRT-PCR assay. The primers G0587 (5′-CGAAACTCTCTCATACCTTCCCGA-3′) and G0588 (5′-CATGGTCCAGTATCTGCCGTCATA-3′) were used for RBCS gene qRT-PCR assay.
4
Modeling Acute Hepatic Injury in Mice
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To mimic acute hepatic injury, C57/BL6J mice were intraperitoneally injected with 10 mg/kg LPS. Mice were sacrificed after injection at 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours. For the blocking experiment, 2 μg/g anti-mouse IL-6 antibody (504506, BioLegend, USA) was intravenously injected into the tail simultaneously with LPS injection, while the control group was intravenously injected with 2 μg/g rat IgG1 (400432, BioLegend, USA). For the inhibition experiment, mice were intragastrically filled with 30 mg/kg ruxolitinib (S1378, SelleckChem, USA) simultaneously with LPS injection, while the control group was intragastrically filled with PBS-diluted DMSO. Mice were sacrificed at 6 hours and 12 hours after injection. Part of the liver tissue was stored in a -80°C freezer for RNA extraction. Liver tissue was ground in liquid nitrogen. Then, RNA was extracted with the TRIzol reagent (15596018, Invitrogen, USA). Reverse transcription was performed by using the Transcriptor cDNA Synth. Kit (4897030001, Roche, USA). With GAPDH as the housekeeping gene, the mRNA levels of IL-6 and GP73 were quantitatively measured by RT-PCR using the FS Universal SYBR Green Master Mix (4913914001, Roche, USA). The primers are listed in Supplementary Table 1 .
5
Quantitative Analysis of mRNA Expression
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Total RNA was extracted from 10 nM dihydrotestosterone (DHT)-treated or equivalent ethanol-treated cells for the indicated times using TRIzol Reagent (15596-018, Invitrogen Corporation, USA) according to the manufacturer’s instructions. For reverse transcription, 1 μg of total RNA was used with a Transcriptor First Strand cDNA Synthesis Kit (04379012001, Roche Applied Science, USA) according to the manufacturer’s procedure. mRNA expression was analyzed by quantitative reverse transcription-PCR (RT-qPCR) using FS Universal SYBR Green Master Mix (4913850001, Roche Applied Science, USA). The following primers were applied in this study:
ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) was used to perform qPCR. Three independent samples were collected in triplicate and analyzed using 2-ΔΔCt relative quantitative analysis.
ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) was used to perform qPCR. Three independent samples were collected in triplicate and analyzed using 2-ΔΔCt relative quantitative analysis.
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