The Total RNA Extraction Kit (Solarbo, Beijing, China) was used to isolate the total RNA. First-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, United States) and Premix Ex Taq SYBR Green PCR kit (Takara, Dalian, China) were used according to the manufacturer’s protocols for reverse transcription and quantitative PCR, respectively. The primers were synthesized as follows: KIF20A, forward: 5′-TGCTGTCCGATGACGATGTC-3′ and reverse: 5′-AGGTTCTTGCGTACCACAGAC-3'; GAPDH, forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3'.
Premix ex taq sybr green pcr kit
Manufactured by Takara Bio
Sourced in Japan
The Premix Ex Taq SYBR Green PCR Kit is a real-time PCR reagent that contains a SYBR Green-based master mix for efficient and sensitive quantification of DNA targets. The kit includes a pre-made reaction mix that simplifies PCR setup and helps ensure consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Cdna synthesis kit, by Takara Bio (4 mentions)
Trizol, by Merck Group (4 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Anti trem 1 primary antibody, by R&D Systems (2 mentions)
Monoclonal rabbit antibodies against sirt6, by Cell Signaling Technology (2 mentions)
Cd42 fitc, by BD (2 mentions)
Human ldl, by Merck Group (1 mentions)
Recombinant human granulocyte macrophage colony stimulating factor gm csf, by R&D Systems (1 mentions)
Cd14 fitc, by BD (1 mentions)
Cd83 fitc, by BD (1 mentions)
Interleukin 4 il 4, by R&D Systems (1 mentions)
Hla dr pe antibodies, by BD (1 mentions)
Trem 1 sirna, by Thermo Fisher Scientific (1 mentions)
Cd1a fitc, by BD (1 mentions)
Cd86 fitc, by BD (1 mentions)
Cd40 fitc, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Becn1, by Cell Signaling Technology (1 mentions)
Lamp2, by Cell Signaling Technology (1 mentions)
6 protocols using premix ex taq sybr green pcr kit
1
Quantifying Gene Expression via RT-qPCR
2
Total RNA Extraction and RT-PCR Analysis
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After total RNA was extracted with a Total RNA Extraction kit (Solarbo, Beijing, China), reverse transcription was conducted using a first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. RT-PCR was then performed using the Premix Ex Taq SYBR Green PCR kit (TaKaRa, Dalian, China), following the manufacturer’s instructions. The primers used in this experiment are summarized in Supplement Table 1
3
Optimizing TREM-1 Silencing in Macrophages
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Lipofectamine® 2000 Transfection Reagent and TREM-1 siRNA were purchased from Invitrogen (Carlsbad, USA). TRIzol, LPS, and human LDL were purchased from Sigma-Aldrich (St. Louis, USA). LP17 (LQVTDSGLYRCVIYHPP) and a sequence-scrambled control-peptide (TDSRCVIGLYHPPLQVY) were produced by GL Biochem (Shanghai) LTD, as described by Gibot et al. [18 (link)]. The peptides were obtained with good yields (>99%), purified, and confirmed by preparative chromatography. These peptides were free of endotoxin. The anti-CD14 and CD4 magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The ELISA kits, CD1a-FITC, CD40-FITC, CD86-FITC, CD83-FITC, CD14-FITC, and HLA-DR-PE antibodies were purchased from BD (Franklin Lakes, USA). The anti-TREM-1 primary antibody and Human sTREM-1 ELISA kit were purchased from R&D (HK, China). The anti-SOCS1and GAPDH primary antibodies were purchased from Cell Signaling (Denver, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). Recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) were purchased from R&D Systems (Minneapolis, USA).
4
Investigating Cellular Mechanisms in Disease
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TRIzol was purchased from Sigma (St Louis, Missouri, USA). TMR red-labeled In-Situ Cell Death Detection reagent and sTREM-1, the sSirt6 enzyme-linked immune sorbent assay (ELISA) kit were purchased from Roche Applied Science (Indianapolis, USA). Monoclonal antibodies against CD62-PE, CD31-PE, CD42-FITC, and IgG-PE were purchased from BD (Shanghai, China), and monoclonal rabbit antibodies against Sirt6, TREM-1, LC3, BECN1, LAMP2, p62, Caspase-1, and GAPDH were purchased from Cell Signaling (Denver, Colorado, USA). Fetal bovine serum (FBS) and Lipofectamine® 2000 Transfection Reagent were purchased from Invitrogen (Carlsbad, CA, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). The adv-Sirt6, LV-Sirt6 shRNA, adv-TREM-1, LV-TREM-1 shRNA, LV-LC3 shRNA, and GFR, GRP double labeled adv-LC3 were purchased from HanBio. (Shanghai, China). Other unmentioned reagents were purchased from Shenggong Bio. (Shanghai, China).
5
Sirt6 Regulation of Endothelial Function
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Glucose and TRIzol were purchased from Sigma (St Louis, Missouri, USA). Monoclonal antibodies against CD62-PE, CD31-PE, CD42-FITC and IgG-PE were obtained from BD (Shanghai, China), and the monoclonal rabbit antibodies against Sirt6, p-eNOS, eNOS and β-actin were obtained from Cell Signaling (Denver, Colorado, USA). The fetal bovine serum (FBS), DMEM culture medium with or without glucose, Lipofectamine® 2000 Transfection Reagent and ECMatirm™ solution were purchased from Invitrogen (Carlsbad, CA, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). The adv-Sirt6, Sirt6 siRNA, CMV-Sirt6 shRNA, Sirt1 siRNA and Drosha siRNA were obtained from HanBio. (Shanghai, China). Other unmentioned reagents were purchased from Shenggong Bio. (Shanghai, China).
6
TREM-1 Silencing in Dendritic Cells
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Lipofectamine® 2000 TransfectionReagent and TREM-1 siRNAwere purchased from Invitrogen (Carlsbad, USA). Trizol was purchased from Sigma (St. Louis, USA).LP17 (LQVTDSGLYRCVIYHPP) and a sequencescrambled control-peptide (TDSRCVIGLYHPPLQVY) were produced by GL Biochem Shanghai LTD, as described by Gibot etal [18] .These peptides were obtained with good yields (>99%), puri ed and con rmed by preparativechromatography, and free of endotoxin.The anti-CD14 magnetic bead was purchased from Merck Millipore(Darmstadt, Germany). IL-4 and GM-CSF were from Peprotech (New Jersey, USA). The ELISA kits and CD1a-FITC, CD40-FITC, CD86-FITC, HLA-DR-PE antibodies were purchased fromBD (Franklin Lakes, USA). The anti-TREM-1 primary antibody was purchased from R&D (HK, China). The anti-SOCS1, GAPDH primary antibodieswere purchased from Cell Signaling (Denver, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCRKit were purchased from Takara (Shiga, Japan). Other reagents were purchased from Gibco(Grand Island, USA).
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