Transcript expression pattern from total RNA isolated from ARPE-19 cells (Qiagen, Hilden, Germany) was determined by RT-PCR using Q5 high GC protocols (New Engand Biolabs, Ipswich, MA, USA). Transcript levels from total RNA extracted from posterior eye segments were determined by SYBR green quantitative real-time RT-PCR (qPCR). All qPCR reagents and PrimePCR primer pairs from BioRad Laboratories (Hercules, CA, USA). Data increase or decrease (fold change) of HIF-regulated transcripts was determined relative to CMX control CNV-induced mice of each corresponding day (ΔΔCt method), after normalization to two housekeep genes.
Qpcr reagents
Manufactured by Bio-Rad
Sourced in United States
QPCR reagents are a set of chemical components used in quantitative polymerase chain reaction (qPCR) analysis. The core function of QPCR reagents is to facilitate the detection and quantification of specific DNA or RNA sequences in a sample through real-time PCR amplification and fluorescent signal measurement.
Automatically generated - may contain errors
Lab products found in correlation
Primepcr primer pairs, by Bio-Rad (1 mentions)
Genistein, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Daidzein, by Merck Group (1 mentions)
Fugene 6 transfection agent, by Promega (1 mentions)
Dual luciferase reporter dlr assay system, by Promega (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Adipored assay reagent, by Lonza (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Rt pcr reagents, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using qpcr reagents
1
ARPE-19 Transcriptome Analysis by RT-qPCR
2
Adipogenic Differentiation Assay
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Quantitative polymerase chain reaction (qPCR) reagents were obtained from BIO-RAD. Rosiglitazone, genistein, and daidzein were purchased from Sigma. The -specific antagonist GlaxoWellcome9662 (GW9662) was a gift from GlaxoSmithKline Pharmaceuticals. AdipoRed assay reagent was bought from Lonza, and Hoechst 33342 from Sigma. Adipocyte differentiation kits (Adipogenesis Assay Kit item no. 10006908) were purchased from Cayman Chemical Company. Fugene 6 transfection agent and the Dual-Luciferase® Reporter (DLR™) assay system were purchased from Promega. qPCR oligos were obtained from Invitrogen.
3
qPCR Primer Design and Reagents
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Primer Express software (Thermo Fisher) was used to design PCR primers which were synthesized by Biosearch Technologies (Petaluma, CA). The gene names, GenBank accession numbers, amplicon sizes, and the sequences (5′ to 3′) of the forward primers and reverse primers, respectively, are described previously38 (link). Chemicals were from Sigma. Cell culture reagents were from Gibco BRL (Thermo Fisher). qPCR reagents were from Bio-Rad.
4
Quantifying Inflammatory Cytokine Expression
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The TRIzol reagent was purchased from Invitrogen, the RT-PCR reagents were purchased from Promega (Madison, WI, USA), and the qPCR reagents were purchased from Bio-Rad. Total RNA was isolated using the TRIzol reagent according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed using M-MLV reverse transcriptase to produce cDNA. qPCR was performed using a Mastercycler with specific primers, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers (GenScript Co., Ltd., Nanjing, China) used in the study are the following: TNF-α, 5′-GCG ACG TGG AAC TGG CAG AAG-3′ (forward) and 5′-TCC ATG CCG TTG GCC AGG AGG-3′ (reverse); IL-1β, 5′-TCT CAT TGT CTC GGT GCT C-3′ (forward) and 5′-CTT TCG GGA AGA GGT TTC A-3′ (reverse); and GAPDH, 5′-CAC CAT CTT CCA GGA GCG AG-3′ (forward) and 5′-GCA GGA GGC ATT GCT GAT-3′ (reverse). The relative levels of the mRNAs were then determined through the 2−ΔΔCt method using GAPDH as the internal control.
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