Absolute qpcr sybr green capillary mix ab gene system

For the measurement of mRNA levels of genes of interest, total RNAs were isolated using Qiagen lysis solution (Qiagen, Maryland, USA) according to the manufacturer's instructions. cDNA was then synthesized via reverse transcription of 1 μg of total RNA. Real time-PCR amplification was then performed with a Roche Light Cycler 2.0 (Mannheim, Germany) using the absolute QPCR SYBR green capillary mix AB gene system (Thermo scientific, UK) essentially as described previously [23 (link)]. The primer sequences used for amplification of target human genes are listed in Table 1.

For the measurement of mRNA levels of target genes, total RNAs were isolated by Qiagen lysis solution (Qiagen, Maryland, USA) according to the manufacturer's instruction. Isolated total RNA was reverse transcribed into cDNA using Go Script reverse transcription system (Promega). Quantitative Real time-PCR was then performed using Light Cycler 2.0 (Mannheim, Germany) with the use of absolute QPCR SYBR green capillary mix AB gene system (Thermoscientific, UK) at 95°C for 15 s, 56°C for 30 s, and 72°C for 45 s. The amount of target mRNA was analyzed via comparative threshold (Ct) method after normalizing target mRNA Ct values to that for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ΔCt). The primer sequences used for amplification of target genes are listed in Table 1.

Total RNA was extracted using Qiagen lysis reagent (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using the Go Script reverse transcription system (Promega Corporation, Madison, WI, USA). Quantitative real-time PCR amplification was then carried out with a Roche LightCycler 2.0 (Mannheim, Germany) using the absolute qPCR SYBR green capillary mix AB gene system (Thermo scientific, UK) at 95 °C for 15 min followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The primer sequences used for amplification of target genes were as follows; SESN2: Forward 5′-CCTTCTCCACACCCAGACAT-3′, Reverse 5′-GTGCATGGCGATGGTGTTAT-3′; pro-IL-1β: Forward 5′-GCCCTAAACAGATGAAGTGCTC-3′, Reverse 5′-GAACCAGCATCTTCCTCAG-3′; GAPDH: Forward 5′-ACCACAGTCCATGCCATCAC-3′, Reverse 5′-TCCACCACCCTGTTGCTGTA-3′.