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Lightcycle 480 sybr green 1 master mix

Manufactured by Roche
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The LightCycle 480 SYBR Green I Master Mix is a ready-to-use solution for real-time PCR amplification and detection using the SYBR Green I dye. It contains all the necessary components, including the DNA polymerase, nucleotides, and SYBR Green I, for efficient and specific real-time PCR reactions.

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Lab products found in correlation

Lightcycle 480 relative quantification software, by Roche (4 mentions) Lightcycle 480 real time pcr system, by Roche (3 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Gapdh, by Abcam (3 mentions) Trizol reagent, by Thermo Fisher Scientific (3 mentions) Tauroursodeoxycholic acid, by Merck Group (2 mentions) Cholic acid, by Merck Group (2 mentions) Taurocholic acid, by Merck Group (1 mentions) Antibody against gapdh, by Santa Cruz Biotechnology (1 mentions) Tauro α muricholic acid, by Merck Group (1 mentions) Midazolam, by Merck Group (1 mentions) Pentoxyresorufin, by Merck Group (1 mentions) Ab137015, by Abcam (1 mentions) Ab181602, by Abcam (1 mentions) Sc 30621, by Santa Cruz Biotechnology (1 mentions) Testosterone, by Merck Group (1 mentions) Diclofenac, by Merck Group (1 mentions) Taurodeoxycholic acid, by Merck Group (1 mentions) Trizol solution, by Thermo Fisher Scientific (1 mentions) Phospho jnk p jnk, by Cell Signaling Technology (1 mentions)

10 protocols using lightcycle 480 sybr green 1 master mix

1

Quantitative Gene Expression Analysis of MAN1A1

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Total RNA extraction and cDNA conversion were performed as described previously [25 (link)]. PCR reactions were performed using 40 ng cDNA, 0.4 µM of the specific forward and reverse primers for MAN1A1 (5′-GTGGACAGTGGGGTCAACAT-3′ and 5′- GCTGCTAGACTTGCGGATCA-3′) in a total of 10 µl LightCycle 480® SYBR green I master mix (Roche Diagnostic, Mannheim, Germany), using the LightCycle 480® real-time PCR system (Roche Diagnostic). β-actin expression was analyzed as an internal control. Gene expression levels were determined using LightCycle 480® Relative Quantification software (Roche Diagnostic).
Phoomak C., Silsirivanit A., Park D., Sawanyawisuth K., Vaeteewoottacharn K., Wongkham C., Lam E.W., Pairojkul C., Lebrilla C.B, & Wongkham S. (2018). O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1. Oncogene, 37(42), 5648-5665.
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2

Bile Acid-Mediated Liver Regulation

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PFDA (98 %), propylene glycol, cholic acid (CA), taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), and tauro-α-muricholic acid (TαMCA) were purchased from Sigma-Aldrich (St Louis, MO). TRIzol was obtained from Invitrogen (CA, USA). Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) assay kits were from Yonghe Sunshine Technology (Changsha, China). Antibodies against total-JNK (t-JNK) and phospho-JNK (p-JNK), total-ATF2 (t-ATF2) and phospho-ATF2 (p-ATF2), NFκB subunit p65, and active form p-p65 were purchased from Cell Signaling Technology (Danvers, USA). Antibody against GAPDH was acquired from Santa Cruz Biotechnologies (CA, USA). Ultrapure water was freshly prepared using a Milli-Q50 SP Water System (MA, USA). LightCycle 480 SYBR Green I Master Mix was obtained from Roche Diagnostics (Mannheim, Germany). All the other chemicals were of the highest grade available from commercial sources.
Luo M., Tan Z., Dai M., Song D., Lin J., Xie M., Yang J., Sun L., Wei D., Zhao J., Gonzalez F.J, & Liu A. (2016). Dual action of peroxisome proliferator-activated receptor alpha in perfluorodecanoic acid-induced hepatotoxicity. Archives of toxicology, 91(2), 897-907.
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3

CYP450 Enzyme Activity Evaluation

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Gemfibrozil was obtained from Hunan Qianjin Xiangjiang Pharmaceutical Co. Ltd (Zhuzhou, China). Trizol was obtained from Invitrogen (CA, USA). The reverse transcription kit was purchased from Thermo Fisher (PA, USA). The LightCycle 480 SYBR Green I Master Mix was purchased from Roche. Antibodies against CYP3A (sc-30621) and CYP2B1/2 (sc-73546) were products by Santa Cruz Biotechnologies (CA, USA). Antibody against GAPDH (ab181602), CYP2C (ab137015) were obtained from Abcam (Cambridge, UK). Midazolam, testosterone, pentoxyresorufin, diclofenac were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). 1′-hydroxy-Midazolam (1-OH-M), 6β-hydroxyl-testosterone (6β-OH-T), resorufin (Reso), and 4-hydroxy-diclofenac (4-OH-D) were obtained from Toronto Research Chemical Inc. (Toronto, ON, Canada). Purified water was freshly prepared using a Millipore Elix (Bedford, USA). All the other chemicals were of the highest grade from commercial sources.
Shi C., Min L., Yang J., Dai M., Song D., Hua H., Xu G., Gonzalez F.J, & Liu A. (2017). PPARα Activation Suppresses Cytochrome P450 Induction Potential in Mice Treated with Gemfibrozil. Basic & clinical pharmacology & toxicology, 121(3), 169-174.
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4

Quantifying MAN1A1 Expression by qPCR

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Total RNA extraction and cDNA conversion were performed as described previously.25 PCR reactions were performed using 40 ng cDNA, 0.4 µM of the specific forward and reverse primers for MAN1A1 (5′-GTGGACAGTGGGGTCAACAT-3′ and 5′- GCTGCTAGACTTGCGGATCA-3′) in a total of 10 µl LightCycle 480® SYBR green I master mix (Roche Diagnostic, Mannheim, Germany), using the LightCycle 480® real-time PCR system (Roche Diagnostic). β-actin expression was analyzed as an internal control. Gene expression levels were determined using LightCycle 480® Relative Quantification software (Roche Diagnostic).
Phoomak C., Silsirivanit A., Park D., Sawanyawisuth K., Vaeteewoottacharn K., Wongkham C., Lam E.W., Pairojkul C., Lebrilla C.B, & Wongkham S. (2018). O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1. Oncogene, 37(42), 5648-5665.
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5

Experimental Cholestatic Liver Injury Assay

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α-Naphthylisothiocyanate (ANIT), cholic acid (CA), tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), tauro-ω-muricholic acid (TωMCA), tauro-α-muricholic acid (TαMCA), taurocholic acid (TCA), β-muricholic acid (βMCA) and ω-muricholic acid (ωMCA) were purchased from Sigma-Aldrich (St Louis, MO). Assay kits for the liver injury markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholestasis markers total bile acid (TBA), alkaline phosphatase (ALP), direct bilirubin (DBIL), and total bilirubin (TBIL) were purchased from Ruiyuan Biotechnology (Ningbo, China). TRIzol solution was purchased from Invitrogen (Dalian, China). The reverse transcription kit and LightCycle 480 SYBR Green I Master Mix were purchased from Roche Diagnostics (Shanghai, China). Antibodies against total mitogen-activated protein kinase kinase 4 (t-MKK4), phospho-mitogen-activated protein kinase kinase 4 (p-MKK4), total-JNK (t-JNK), and phospho-JNK (p-JNK) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against NF-κB subunit p65, phospho-p65 (p-p65), total STAT3 (t-STAT3), phospho-STAT3 (p-STAT3), and GAPDH were acquired from Abcam.
Dai M., Hua H., Lin H., Xu G., Hu X., Li F., Gonzalez F.J., Liu A, & Yang J. (2018). Targeted Metabolomics Reveals a Protective Role for Basal PPARα in Cholestasis Induced by α-Naphthylisothiocyanate. Journal of proteome research, 17(4), 1500-1508.
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6

Cytokine Receptor Expression Profiling

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Expressions of cytokine receptors were semiquantitatively measured by RT-PCR as previously described [40 (link)]. Total RNA was extracted by the TRIzol™ Reagent (Thermo Fisher Scientific), and cDNA was synthesized by Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. RT-PCR was performed using the LightCycle 480® real-time PCR system (Roche Diagnostic, Mannheim, Germany). Forty nanograms of cDNA, 0.4 μM primers, and LightCycle 480® SYBR green I master mix (Roche Diagnostic) was included. The gene expression level was quantified by LightCycle 480® Relative Quantification software (Roche Diagnostic). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Oligonucleotide primers are listed in Table 1.
Dana P., Kariya R., Lert-itthiporn W., Seubwai W., Saisomboon S., Wongkham C., Okada S., Wongkham S, & Vaeteewoottacharn K. (2021). Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway. International Journal of Molecular Sciences, 22(24), 13496.
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7

Quantitative RT-PCR Protocol for Gene Expression

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Total RNA was extracted using Trizol reagent (Ambion) and converted to cDNA using the high capacity cDNA Reverse Transcription Kit (Applied Biosystems). Reactions were performed using cDNA converted from 40 ng of total RNA, 0.4 μM of forward- and reverse-primers (Supplementary Table S1), and LightCycle 480® SYBR green I master mix (Roche Diagnostic). PCR was performed in the LightCycle 480® real-time PCR system (Roche Diagnostic, Mannheim, Germany) with a slight modification from that previously described40 (link). The annealing at 60 °C for 10 sec and the PCR reaction was ended with a final extension at 72 °C for 10 min. The gene expression level was relatively quantified from duplicated samples using LightCycle 480® Relative Quantification software (Roche Diagnostic). β-2microglobulin (B2M) was used as an internal control41 (link).
Phoomak C., Vaeteewoottacharn K., Sawanyawisuth K., Seubwai W., Wongkham C., Silsirivanit A, & Wongkham S. (2016). Mechanistic insights of O-GlcNAcylation that promote progression of cholangiocarcinoma cells via nuclear translocation of NF-κB. Scientific Reports, 6, 27853.
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8

Anti-Diabetic Compound Screening Protocol

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Gemfibrozil, fenofibrate, Oil Red O, dexamethasone, 3-isobutyl-1- methyl xanthine and bovine insulin were all obtained from Sigma-Aldrich (MO, USA). TC and TG assay kits were purchased from Ruiyuan Biotechnology (Ningbo, China). Antibodies against NFκB subunit p65 and active form phospho-p65 (p-p65), total STAT3 (t-STAT3), phospho-STAT3 (p-STAT3), phospho-JNK (p-JNK), phospho-ATF2 (p-ATF2) and GAPDH were acquired from Abcam (MA, USA). The reverse transcription kit and LightCycle 480 SYBR Green I Master Mix were obtained from Roche Diagnostics (Mannheim, Germany). Blood glucose meter test strips were bought from Johnson & Johnson Pharmaceuticals Ltd (USA). Ultrapure water was freshly prepared using a Milli-Q50 SP Water System (MA, USA). Medium-fat diet (MFD, 31% kcal fat) was obtained from SLACOM (Shanghai, China). All the other chemicals were of the highest grade available from commercial sources.
Hua H., Yang J., Lin H., Xi Y., Dai M., Xu G., Wang F., Liu L., Zhao T., Huang J., Gonzalez F.J, & Liu A. (2018). PPARα-independent action against metabolic syndrome development by fibrates is mediated by inhibition of STAT3 signaling. The Journal of pharmacy and pharmacology, 70(12), 1630-1642.
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9

Gemfibrozil and Fenofibrate Pharmacological Evaluation

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Gemfibrozil was obtained from Hunan Qianjin Xiangjiang Pharmaceutical Co. Ltd. (Zhuzhou, China). Fenofibrate was purchased from Shangqiu Chemry Chemicals Co. Ltd. (Shangqiu, China). All the ELISA kits were purchased from B&D Systems (Shanghai, China). The reverse transcription kit was provided by Thermo Fisher (Pennsylvania, USA). The LightCycle 480 SYBR Green I Master Mix was purchased from Roche. Purified water was freshly prepared using a Millipore Elix (Massachusetts, USA) system. All the other chemicals were of the highest grade available from commercial sources.
Song D., Luo M., Dai M., Bu S., Wang W., Zhang B., Gonzalez F.J, & Liu A. (2016). PPARα-dependent increase of mouse urine output by gemfibrozil and fenofibrate. Canadian journal of physiology and pharmacology, 95(2), 199-205.
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10

Hepatoprotective Mechanisms of ANIT

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Chemicals and reagents. ANIT was purchased from Sigma-Aldrich; Merck KGaA. Alkaline phosphatase (ALP), total bile acid (TBA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) assay kits were purchased from Ningbo Ruiyuan Biotechnology co., Ltd. TRIzol ® and the reverse transcription kit were purchased from Invitrogen (Thermo Fisher Scientific, Inc). The LightCycle 480 SYBR Green I Master Mix was obtained from Roche diagnostics. Antibodies against phosphorylated (p)-MAPK kinase 4 (MKK4) (1:1,000; product no. 4514) and total (t)-MKK4 (1:1,000; product no. 9152), t-JNK (1:1,000; product no. 9252) and p-JNK (1:1,000; product no. 9912), p-c-Jun (1:1,000; product no. 2361) and t-c-Jun (1:1,000; product no. 9165), were obtained from cell Signalling Technology, Inc. Primary antibodies against cYP7A1 (1:1,000; product code ab65596), cYP8B1(1:1,000; product code ab191910), GAPdH (1:10,000; product code ab181602) were acquired from Abcam. Secondary antibody IgG H&L (HRP) was also obtained from Abcam (1:2,000; product code ab205718). All the other chemicals were of the highest grade available from commercial sources.
Zheng X., Dai M., Xu G., Chang L., Yang J, & Liu A. (2020). Inhibition of inflammation by SP600125 in cholestatic liver injury is dependent on the administration‑based exposure profile. International journal of molecular medicine, 46(6).
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