Tnf alpha human elisa kit ultrasensitive

From all study participants, 5 ml of whole blood was obtained (stored in − 80 °C). DNA Blood Mini Kit (Qiagen, Canada) was used to DNA isolation. DNA quality and quantity were evaluated with the use of NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, USA). Genotyping was performed using a real-time PCR method with allele-discriminating software. The Genotyping Master Mix and TaqMan probes (Applied Biosystems, USA) specific for studied TNF-α SNP (Thermo Fisher Scientific, USA, SNP ID: C_7514871_10, cat. no. 4351379) were used to DNA amplification under the condition of manufacturer protocol provided with kit in RT7500 Real-time PCR device (Applied Biosystems, USA). Because we were interested in the assessment of the generalised inflammation process (TNF-α in circulation) instead of local changes (TNF-α in saliva), we decided to use plasma instead of saliva as a test material. The plasma TNF-α level was assessed using TNF alpha Human ELISA Kit Ultrasensitive (Thermo Fisher Scientific, USA, cat. no. KHC3014). The detection range was 0.2–32 pg/mL, and the sensitivity was equal to the minimal detectable dose of this kit (< 0.09 pg/mL).

miRNA was isolated from 250 µL of plasma using miRNeasy serum/plasma kit (Qiagen, Hilden, Germany) and then reversely transcribed into complementary DNA (cDNA) by TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Amplification of cDNA was conducted in StepOnePlus Real-Time PCR device (Applied Biosystems, Foster City, CA, USA) with the use of TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, USA) and ready to use fluorescently labeled probes (TaqMan probes dedicated for circulating miRNA analysis) targeting miRNA-130a and miRNA-26a (internal control) (Thermo Fisher Scientific). All steps of miRNA analysis were conducted under the conditions of protocols provided by the manufacturer. The level of miRNA-130a expression was normalized in relation to miRNA-26a expression using ΔCt, 2^−ΔCt and 2^−ΔΔCt formula.
Plasma TNF-α level was measured using TNF alpha Human ELISA Kit Ultrasensitive (Thermo Fisher Scientific). The detection range was 0.2–32 pg/mL and the sensitivity was equal to the minimum detectable dose of this kit (<0.09 pg/mL).

DNA was isolated from whole blood samples using DNA Blood Mini Kit (Qiagen, Canada). Genotyping was conducted using real-time PCR method and TaqMan SNP genotyping assay with allele discriminating software. The TaqMan fluorescently labeled probes (Applied Biosystems, USA) targeting the studied TNF-α SNP and Genotyping Master Mix (ThermoFisherScientific, USA) were used for DNA amplification in the StepOnePlus Real-Time PCR System (Applied Biosystems, USA). All genotyping steps were conducted under the conditions of protocol provided by the manufacturer.
Plasma TNF-α level was measured using TNF alpha Human ELISA Kit Ultrasensitive (Thermo Fisher Scientific, USA). The detection range was 0.2–32 pg/mL and the sensitivity was equal to the minimal detectable dose of this kit (< 0.09 pg/mL).