Qpcr sybr green master mix

We collected the following data via medical records and questionnaire investigation: age, gender, family history, feeding type (cow milk, breast milk, mixed), parent smoking, keeping pet, history of diseases, growth and development status, history of allergic. The questionnaire was screened according to Williams diagnostic criteria, and the patients with suspected AD were verified, diagnosed and counted by specialists according to the diagnostic criteria. IgE expression of cord blood was detected using immunofluorescence analysis, and interleukin-4, 5,10 were measured using the double antibody sandwich ELISA technique. The mRNA expression of VDR was detected RT-PCR with Hifair^TM first‐strand cDNA synthesis SuperMix and qPCR SYBR Green Master Mix (Yesen, Shanghai, China) using peripheral blood mononuclear cell: a plastic blood bag containing a blood sample into a centrifuge tube adapter, trim after centrifugation, remove the upper plasma layer will be removed in the middle of shallow to shallow layer was removed diluted with normal saline was added; the resulting product into a liquid containing lymphocyte separation centrifuge tube, wherein the product obtained is located in the upper lymphocyte separation medium; centrifugation, draw intermediate buffy coat washed, centrifuged supernatant was removed to obtain peripheral blood PBMC.

Total RNA was isolated from whole blood from the AR patients and healthy controls using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. VDR gene (Fok I) expression was determined using RT‐PCR with Hifair^TM first‐strand cDNA synthesis SuperMix and qPCR SYBR Green Master Mix (Yesen, Shanghai, China). The 2−ΔΔCT method was used to calculate relative expression.