Total RNA was extracted from TM cells from mock-infected or infected with HSV-1 at a MOI 1 of 1 and/or stimulated with recombinant active TGF-β1 at 15 ng/ml (0.6 nM) for 12 h using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA was reverse transcribed using a cDNA synthesis kit (PrimeScript RT Reagent Kit, Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions. Using a Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche, Mannheim, Germany), the relative expression levels of mRNA were determined as previously described.[10 (link)] Reactions for each sample were run in triplicate, cycle thresholds were normalized to β-actin expression, and comparative quantitation was performed (LightCycler software, version 4.1, Roche). Only individual PCR samples with single peak dissociation curves were selected for data analysis. Table 1 shows the sequences of the primer sets. To ensure equal loading and amplification, all products were normalized to a β-actin as an internal control. The results are the averages of three independent experiments.
Diagnostics lightcycler 2.0 real time pcr system
Manufactured by Roche
Sourced in Germany
The Roche Diagnostics LightCycler 2.0 Real-Time PCR System is a laboratory instrument used for real-time polymerase chain reaction (PCR) analysis. It is designed to amplify and detect specific DNA sequences in real-time. The system includes a thermal cycler, optics for fluorescence detection, and software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Lightcycler software, by Roche (1 mentions)
Mcp 1 elisa kit, by R&D Systems (1 mentions)
Pre coated plates, by R&D Systems (1 mentions)
Sensifast sybr hi rox kit, by Meridian Bioscience (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
4 protocols using diagnostics lightcycler 2.0 real time pcr system
1
Quantitative Real-Time PCR Analysis of HSV-1 and TGF-β1 Effects
2
Quantitative Analysis of Transcriptional Responses
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From the mock‐infected or HSV‐1 infected TM cells with or without various treatments, total RNA was extracted using an RNeasy Mini Kit (Qiagen). The total RNA was quantified and reverse‐transcribed to cDNA using PrimeScript RT reagent Kit (Takara Bio). The relative expression levels of mRNAs were determined using a Roche Diagnostics LightCycler 2.0 Real‐Time PCR System (Roche). The sequences of the real‐time PCR primer pairs are shown in Table 1 . To ensure equal loading and amplification, all products were normalized relative to β‐actin transcript as an internal control.
Levels of secreted monocyte chemotactic protein (MCP)‐1 were measured using a commercially available MCP‐1 ELISA Kit with pre‐coated plates (R&D Systems). Conditioned medium was harvested, cleared by centrifugation and stored at –70℃. The medium was subsequently acid‐activated and directly assayed using an ELISA plate reader at 450 nm in accordance with the manufacturer's instructions. Protein concentrations were calculated from a standard curve with twofold serial dilutions with the highest standard of 500 pg/ml.
Levels of secreted monocyte chemotactic protein (MCP)‐1 were measured using a commercially available MCP‐1 ELISA Kit with pre‐coated plates (R&D Systems). Conditioned medium was harvested, cleared by centrifugation and stored at –70℃. The medium was subsequently acid‐activated and directly assayed using an ELISA plate reader at 450 nm in accordance with the manufacturer's instructions. Protein concentrations were calculated from a standard curve with twofold serial dilutions with the highest standard of 500 pg/ml.
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from tissues of the lung, liver, and kidney using an RNeasy Plus Mini Kit (QIAGEN GmbH, Hilden, Germany). RNA was reverse transcribed using a PrimeScript RT Reagent Kit (TaKaRa Bio Inc., Shiga, Japan). Relative expression levels of mRNA were determined using a Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with a SensiFAST™ SYBR® Hi-ROX Kit (Bioline, Luckenwalde, Germany) and gene-specific primers (Table 1 ). A total volume of 10 μL of reaction system liquid was subjected to the following PCR program: 1 cycle at 95°C for 30 seconds (initial denaturation), followed by 41 cycles at 95°C for 5 seconds, 60°C for 25 seconds. All protocols were performed according to the manufacturer's instructions.
4
Quantitative Retinal Gene Expression Analysis
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Total RNA was extracted from neurosensory retinas using the RNeasy Mini Kit (Qiagen, Valencia, CA). The RNA was reverse transcribed using a cDNA synthesis kit (PrimeScript RT Reagent Kit; Takara Bio, Kusatsu, Japan) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed using TOPreal qPCR 2Â PreMIX (Enzynomics, Daejeon, Korea) on a Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche, Mannheim, Germany). Reactions for each sample were run in triplicate, cycle thresholds were normalized to GAPDH expression, and comparative quantitation was performed using LightCycler 167 software version 4.1 (Roche). qPCR was performed using primers for glutamateammonia ligase (GLUL), also known as glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP), known glial cell activation markers 45, 46 ; tumor necrosis factor (TNF ), also known as tumor necrosis factor alpha (TNF-a) which induces expression of other inflammatory markers 47, 48 ; TXNIP, which links oxidative stress to inflammation and apoptosis in T2D 12, 13, 49, 50 ; and VEGF, an angiogenic factor induced in retinal ischemia and in inflammatory processes in T2D. 12, 18, 23, 51, 52 Primer sequences are listed in Table 1.
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