Universal probes one step pcr kit

RNA used for qRT-PCR and RNAseq was isolated from a 4-mm biopsy specimen of the gastric corpus lesser curvature. RNA was extracted in TRIzol (Thermo Fisher Scientific) and precipitated from the aqueous phase using 1.5 volumes of 100% ethanol. The mixture was transferred to a RNeasy column (Qiagen, Hilden, Germany), and the remaining steps were followed according to the RNeasy kit manufacturer’s recommendations. RNA was treated with RNase-free DNase I (Qiagen) as part of the isolation procedure. Reverse-transcription followed by qPCR was performed in the same reaction using the Universal Probes One-Step PCR kit (Bio-Rad Laboratories, Hercules, CA) and the TaqMan primers (Thermo Fisher Scientific) Cftr (Mm00445197_m1), Olfm4 (Mm01320260_m1), Wfdc2 (Mm00509434_m1), Zfp36 (Mm00457144_m1), Il33 (Mm00505403_m1), and Il13 (Mm00434204_m1) (Thermo Fisher Scientific) on a Quantstudio 6 (Thermo Fisher Scientific). mRNA levels were normalized to the reference gene Ppib (Mm00478295_m1).