Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). qRT-PCR assays for miRNAs were performed using miScript SYBR Green PCR kit (Qiagen, Valencia, CA), whereas those for mRNAs were done using LightCycler® DNA master SYBR Green-I kit (Roche, Mannheim, Germany) according to the manufacturer's instruction.
Lightcycler dna master sybr green 1 kit
Manufactured by Roche
Sourced in Germany, Switzerland
The LightCycler DNA Master SYBR Green I Kit is a real-time PCR reaction mix designed for use with the LightCycler system. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, allowing for the quantification of DNA targets during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Lightcycler system, by Roche (5 mentions)
Lightcycler 480 system, by Roche (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Lc480 lightcycler system, by Roche (3 mentions)
Lightcycler 2, by Roche (3 mentions)
Miscript sybr green pcr kit, by Qiagen (2 mentions)
Lightcycler 1, by Roche (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Reverse transcription pcr kit, by Promega (2 mentions)
Lightcycler, by Roche (2 mentions)
Sensifast cdna synthesis kit, by Meridian Bioscience (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Abi 7900ht real time pcr system, by Thermo Fisher Scientific (2 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
Reverse transcription system, by Qiagen (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Cell cycle detection kit, by Beyotime (1 mentions)
36 protocols using lightcycler dna master sybr green 1 kit
1
RNA Extraction and qRT-PCR Analysis
2
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from HaCaT, SZ95 cells or mouse skin using TRIzol reagent (Invitrogen). cDNA was synthesized from 1000 ng of total RNA using a reverse transcription system (Qiagen, Hilden, Germany).
The cDNA was used as the template for qRT-PCR. qRT-PCR was carried out with the LightCycler-DNA Master SYBR Green I kit (Roche Molecular Biochemicals). The primer sets for TLR-2, TNF-α, IL-8, IL-6, IL-1β, and GAPDH were purchased as the QuantiTect primer assay (Qiagen). GAPDH was used as an endogenous control.
The cDNA was used as the template for qRT-PCR. qRT-PCR was carried out with the LightCycler-DNA Master SYBR Green I kit (Roche Molecular Biochemicals). The primer sets for TLR-2, TNF-α, IL-8, IL-6, IL-1β, and GAPDH were purchased as the QuantiTect primer assay (Qiagen). GAPDH was used as an endogenous control.
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated employing Trizol reagent (Invitrogen, Carlsbad, CA) and subjected to reverse transcription. The resulting cDNA was subjected to amplification through qRT-PCR utilizing the LightCycler DNA Master SYBR Green-I Kit (Roche, Mannheim, Germany), as instructed by the manufacturer’s guidelines. Gapdh and β-actin served as normalization control. The primer sequences used for qRT-PCR assays are listed in Supplementary Table 1 .
4
Gene Expression Quantification by qPCR
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For quantification of gene expression qPCR was performed using the LightCycler System LC480 and LightCycler-DNA Master SYBR Green I Kit (Roche, Mannheim, Germany) as described previously46 (link). Primer sequences are available on request. Gene expression was calculated by the delta-delta CT method using 36B4 as a reference gene47 . Relative gene expression was calculated by setting the mean of the euthyroid control group to 1 and then calculating each individual value of the three groups of mice studied.
5
Quantitative Real-Time PCR Analysis of Tyrosine Hydroxylase Expression
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Total RNA was extracted from the CeA tissue samples using a Trizol reagent (Invitrogen, Carlsbad, CA) and the cDNA was synthesized by reverse transcription using an oligo (dT) primer. Then, a real-time PCR assay was performed using a LightCycler 1.5 (Roche, Mannheim, Germany) and the LightCycler DNA Master SYBR green-I kit according to the manufacturer's instructions. The primers (Bioneer Corporation, Daejeon, Republic of Korea) were 5′-ATGCCCACCCCCAGCGCCCC-3′ (sense) and 5′-GACACTTTTCTTGGGAACCA-3′ (antisense) for TH and 5′-GTCGTACCACTGGCATTGTG-3′ (sense) and 5′-GCCATCTCTTGCTCGAAGTC-3′ (antisense) for β-actin. The housekeeping gene β-actin was used as an endogenous reference and the relative expression levels of TH mRNA were calculated using the following formula: ΔCT = CT (TH) − CT (β-actin); ΔΔCT = ΔCT (treated) − ΔCT (saline). The levels were expressed as 2−ΔΔCT.
6
Cell Cycle Analysis of HepG2 Cells
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HepG2 (a human HCC cell line) was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Sigma-Aldrich; Merck Milipore, Darmstadt, Germany) and 100 µg/ml streptomycin (Sigma-Aldrich; Merck Milipore) at 37°C, 5% CO2. The Cell Cycle Detection kit was purchased from Beyotime Institute of Biotechnology (Suzhou, China). Anti-p21 [also known as cyclin-dependent kinase inhibitor 1A (CDKN1A)] (bs-10129R), anti-tumor protein p53 inducible protein 3 (TP53I3) (bs-6144R) and anti-wild-type p53-induced phosphatase 1 (Wip1, also known as PPM1D) (bs-2447R) antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The RNeasy Mini kit was purchased from Qiagen GmbH (Hilden, Germany), while First-strand cDNA Synthesis kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The LightCycler DNA Master SYBR Green I kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany).
7
Liver RNA Extraction and RT-qPCR Analysis
8
Quantitative Gene Expression Analysis of CRF, CRFR1, ppN/OFQ, and NOP
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Total RNA was isolated from CeA tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was produced from the RNA with a reverse transcription PCR kit (Promega, Madison WI, USA). Next, qPCR analysis was executed with a LightCycler® DNA Master SYBR Green-I kit (Roche Diagnostics, Mannheim, Germany) using a LightCycler 2.0 (Roche Diagnostics) according to the manufacturer’s instructions. The primers for PCR amplification of CRF, CRFR1, ppN/OFQ, and NOP were as follows: 5′-CTCTCTGGATCTCACCTTCCAC-3′ (sense) and 5′-CTAAATGCAGAATCGTTTTGGC-3′ (antisense) for CRF; 5′-GTCTCCAGG GTCGTCTTCAT-3′ (sense) and 5′-CGGACCTCACTGTTCAGAA-3′ (antisense) for CRFR1; 5′-TGCAGCACCTGAAGAGAATG-3′ (sense) and 5′-CAACTTCCGGGCTGACTTC-3′ (antisense) for ppN/OFQ; 5′-AGCTTCTGAAGAGGCTGTGT-3′ (sense) and 5′-GACCTCCCAGTATGGAGCAG-3′ (antisense) for NOP receptor; and 5′-GTCGTACCACTGGCATTGTG-3′ (sense) and 5′-GCCATCTCTTGCTCGAAGTC-3′ (antisense) for β-actin. The results were normalized to the housekeeping gene β-actin, and relative gene expression was calculated 2−ΔΔCT method with the following formula:
9
Quantitative PCR Analysis of Extracellular Matrix
10
Transcriptomic Analysis via qRT-PCR
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Total RNAs were isolated with Trizol (Invitrogen, Carlsbad, CA, USA) and qRT-PCR assays for mRNAs were performed using LightCycler® DNA master SYBR Green-I kit (Roche, Mannheim, Germany). miRNAs levels were measured using miScript SYBR Green PCR kit (Qiagen) according to the manufacturer's instruction.
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