T-2 toxin was purchased from Puruibang Biological Engineering Co., Ltd. (Qingdao, Shandong, China). BA was purchased from Sigma-Aldrich (St. Louis, MO, USA).VE (Vitamin E) was bought from Sigma-Aldrich (St Louis, MO, USA). Trizol was purchased from Life Technologies (Ambion, Life Technologies Inc., Carlsbad, CA, USA), while the primescript RT reagent kit and SYBR Green I fluorescent dyes were purchased from Takara (Shiga, Japan). A BCA protein assay kit and assay kits for measuring total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH), catalase (CAT), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Biotech (Nanjing, Jiangsu, China). Enhanced chemiluminescence (ECL) reagent was purchased from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, Jiangsu, China). A mouse testosterone ELISA kit was purchased from Wuhan Huamei Biological Engineering Co., Ltd. (Wuhan, Hubei, China). The primary antibodies for β-actin, STAT3, JAK2, Bax, Bcl-2, p-STAT3, p-JAK2, and caspsae-3 were obtained from Cell Signaling Technology (Boston, MA, USA).
Sybr green 1 fluorescent dye
Manufactured by Takara Bio
Sourced in China, Japan
SYBR Green I is a fluorescent dye used in molecular biology applications. It binds to double-stranded DNA and emits a green fluorescent signal upon excitation, allowing the detection and quantification of DNA in various procedures.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Merck Group (3 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
Caspsae 3, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Nanjing Jiancheng (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
He staining solution, by Wuhan Servicebio Technology (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Glutathione (gsh), by Nanjing Jiancheng (1 mentions)
Cat assay kit, by Nanjing Jiancheng (1 mentions)
Abi 7300 thermocycler, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
10 protocols using sybr green 1 fluorescent dye
1
Investigating T-2 Toxin Mitigation Strategies
2
Mycotoxin T-2 Toxin Mitigation
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T-2 toxin was purchased from Pribolab Pte. Ltd. (Singapore). BA was purchased from Sigma (St. Louis, MO, USA). VE, used as positive control [56 (link),57 (link)], was bought from Sigma-Aldrich (St Louis, MO, USA). MDA, GSH, SOD, GSH-PX and CAT assay kits were acquired from Nanjing Jiancheng Biotech (Nanjing, Jiangsu, China). The H&E staining solution was purchased from Servicebio (Wuhan, Hubei, China). IgG, SIgA, IgM, C3, C4 and DAO kits were supplied from Elabscience Biotechnology Co., Ltd. (Wuhan, Hubei, China). SYBR Green I fluorescent dyes and the Primescript RT reagent kit were from Takara (Shiga, Japan) and trizol was supplied by Life Technologies (Carlsbad, CA, USA). The IKB-α antibody, NF-κB p65 antibody and β-actin antibody were bought from Cell Signaling Technology, Inc. (Danvers, MA, USA), and all other reagents were analytical grade.
3
Quantitative Real-Time PCR Analysis of Intestinal Gene Expression
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Quantitative real-time PCR (RT-qPCR) analysis was performed, as described previously [20 (link)]. Briefly, the total RNA of the duodenum, jejunum, and ileum was extracted using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and then reversely transcribed into complementary DNA (cDNA) using RevertAid reverse transcriptase (Takara, Otsu, Japan). RT-qPCR analysis was performed with SYBR green I fluorescent dye (Takara, Otsu, Japan). The relative levels of the mRNA expression of the target genes were calculated using the 2−ΔΔCt method [21 (link),22 (link)]. All primer sequences are presented in Table 2 .
4
RT-PCR protocol for gene expression analysis
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Primers used for RT-PCR were designed by querying PrimerBank with the Gene ID; sequences are listed in Additional file 1: Table 1 . Total RNA was extracted from PK-15 cells using TRIzol reagent (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer's protocol. cDNA of each sample was transcribed by the PrimeScript RT reagent kit (TaKaRa Biotechnology, Beijing, China), and real-time PCR was conducted using SYBR Green I fluorescent dye (TaKaRa Biotechnology, Beijing, China) according to the manufacturer's guidelines.
5
Total RNA Extraction and qRT-PCR in HepG2 Cells
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Total RNA in HepG2 cells was extracted by Trizol reagent (Sigma‐Aldrich) and transcribed to cDNA by PrimeScript RT reagent kit (TaKaRa Biotechnology) according to manufacturer's protocol. Real‐time PCR was performed by the SYBR Green I fluorescent dye (TaKaRa Biotechnology), according to the manufacturer's guidelines. Each sample was assessed in triplicate experiments. The primer sequences (GenScript) used were listed in Table 1 .
6
Extraction and Quantification of miR-21 and Proinflammatory Cytokines
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Total RNA from cells was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc) according to the manufacturer's protocol. The cDNA was reverse transcribed using Prime Script™ RT master mix according to the manufacturer's protocol (Takara Biotechnology Co., Ltd.). The temperature protocol was: 37°C for 15 min; followed by 85°C for 5 sec and 4°C for 20 sec. RT-qPCR was performed on an ABI 7300 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the expression of miR-21 was quantified using SYBR Green I fluorescent dye (Takara Biotechnology Co., Ltd.). The miR-21 expression levels were normalized to the expression of U6. The thermocycling conditions were: 95°C for 2 min; followed by 40 cycles of 95°C for 15 sec and 58°C for 20 sec. The expression levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α were normalized to the expression of GAPDH. The thermocycling conditions were: 95°C for 2 min; followed by 40 cycles of 95°C for 5 sec, 56°C for 20 sec; and 72°C for 25 sec, 65°C for 5 sec, and 95°C for 50 sec. The relative levels of miR-21 or IL-6, IL-1β and TNF-α was calculated using the comparative 2−ΔΔCq method (27 (link)) normalized to U6 or GAPDH, respectively. The sequences of the primers used were presented in Table I .
7
Quantitative Real-Time PCR for ARPC1B Expression
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Total RNA was extracted by using TRIzol Reagent (Invitrogen, United States) according to the manufacturer’s protocol. cDNA was obtained by the reverse transcription system. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green I fluorescent dye (TaKaRa Bio, China) on the LightCycler® 480 Instrument II (Roche, Germany). The relative expression of ARPC1B in each sample was normalized to GAPDH and calculated by using the 2−ΔΔCt method. The primers were described as follows: ARPC1B, gttat ttcga gcagg agaat gac (F), gtagg ctgaa aagat ccgac a (R); GAPDH, gtgga cctga cctgc cgtct (F), ggagg agtgg gtgtc gctgt (R).
8
Quantification of Fibrosis Markers in HSC-LX2 Cells
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Total RNA was extracted from HSC-LX2 cells using Trizol reagent according to the protocol provided by manufacturer (Sigma-Aldrich, St. Louis, MO, USA) and then reversely transcribed to cDNA by PrimeScript RT reagent kit (TaKaRa Biotechnology, Beijing, China). Real-Time PCR was conducted by the SYBR Green I fluorescent dye (TaKaRa Biotechnology, Beijing, China), according to the manufacturer's guidelines. Glyceraldehyde phosphate dehydrogenase (GAPDH) was served as an invariant control, and mRNA levels were expressed as fold changes after normalizing to GAPDH. Results came from triplicate experiments. Table 1 listed out the primers (Genscript, Nanjing, China) used.Table 1
Primers used for determination of mRNA expression levels in human HSC-LX2 cells.
| Gene | Forward sequence | Reverse sequence |
|---|---|---|
| Fibronectin | 5′-AGCCGCCACGTGCCAGGATTAC-3′ | 5′-CTTATGGGGGTGGCCGTTGTGG-3′ |
| α1(I)-procollagen | 5′-AGAGGAAGGAAAGCGAGGAG-3′ | 5′-GGACCAGCAACACCATCTG-3′ |
| α-SMA | 5′-GACAATGGCTCTGGGCTCTGTAA-3′ | 5′-ATGCCATGTTCTATCGGGTACTTCA-3′ |
| GAPDH | 5′-CTTCTTTTGCGTCGCCAGCCGA-3′ | 5′-ACCAGGCGCCCAATACGACCAA-3′ |
9
Liver Tissue RNA Extraction and qRT-PCR
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Total RNA was extracted from liver tissues and LO2 cells by using Trizol reagent according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, United States) and further reverse-transcribed into cDNA by using PrimeScript RT reagent kit (TaKaRa Biotechnology, Beijing, China). Real-time PCR was performed using the SYBR Green I fluorescent dye (TaKaRa Biotechnology, Beijing, China), according to the manufacturer’s protocol. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as an invariant control, and mRNA levels were expressed as fold changes after normalizing to GAPDH. The experiment was performed in triplicates. Primers (Genscript, Nanjing, China) are listed in Table 2 below.
10
Total RNA Extraction and RT-qPCR Analysis
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Total RNA was isolated using Trizol reagent (TaKaRa, Dalian, China). cDNA was synthesized using a Primescript™ RT reagent kit (TaKaRa, Dalian, China). Real-time PCR was performed using the SYBR Green I fluorescent dye (TaKaRa, Dalian, China). The primer pairs were listed in Table S3 .
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