Total mRNA was isolated from the THP-1 cells using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions (Applied Biosystems, Darmstadt, Germany). Real-time PCR was carried out on the 7900HT Fast Real-Time PCR system using TaqMan Gene Expression Assays primer/probe sets (all from Applied Biosystems) and the standard thermal-cycling conditions for relative quantification designed by the manufacturer. Quantification of the PCR signals for each sample was done by comparing the cycle threshold values in duplicate for the gene of interest with the cycle threshold values for the GAPDH housekeeping gene. The mean relative mRNA expression was determined by using SDS software V2.2 (Applied Biosystems).
Taqman gene expression assays primer probe sets
Manufactured by Thermo Fisher Scientific
TaqMan Gene Expression Assays primer/probe sets are pre-designed, gene-specific primer and probe sets for use in real-time PCR-based gene expression analysis. The assays are designed to provide efficient and specific detection and quantification of target gene transcripts.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Sds software v2, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
2 protocols using taqman gene expression assays primer probe sets
1
Real-Time PCR Gene Expression Analysis
2
Quantitative Real-Time PCR Analysis of Lung Gene Expression
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Quantitative real-time PCR (qRT-PCR) was performed on cDNA samples of lung homogenates, as described (Noël et al., 2020 (link)). We used inventoried TaqMan Gene Expression Assays primer-probe sets (Applied Biosystems) and an Applied Biosystems 7300 Real-Time PCR System to evaluate the expression of selected genes. For the asthma study, a total of 24 genes were analyzed by RT-PCR, while for the emphysema study a total of 8 genes were analyzed by RT-PCR. For each gene, we used reaction volumes of 25 μl and 40 reaction cycles. We used the comparative cycle threshold (ΔΔCT) method to determine relative gene expression, with each gene normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt1) expression. Results are reported as fold-change in treated samples compared to controls (2−ΔΔCT).
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