After the total mRNA was isolated, it was reverse-transcribed using the High-Capacity cDNA kit according to the manufacturer’s protocol (Thermo Fisher Scientific, USA). The mRNA and cDNA concentration and quality were determined using a NanoDrop spectrophotometer (Kisker, Germany) and 1% agarose gels, respectively. Samples were stored at -80 ° C until use. cDNA was then amplified by real-time quantitative PCR (qPCR) using TaqMan probes: TTLL12: Hs00209450_m1, CDKN3: Hs00193192_m1, CDC16: Hs00187430_m1, PTPRA: Hs00160751_m1, MZT2B: Hs01117110_sH, UBE2T: Hs00928040_m1 and ACTB (4333762F; Thermo Fisher Scientific, USA) gene was selected as an internal control [27 (link)]. All qPCRs were performed in triplicate in 7500 Fast Real-Time PCR instrument (Thermo Fisher Scientific, USA).
The relative quantification of gene expression was calculated according to the method of Livak and Schmittgen [71 (link)]. We used a control sample of non-neoplastic gastric mucosa cells MNP01 (Normal gastric mucosa cell Line 01) pooled from 10 healthy patients [16 (link)], which was used as a calibrator for each tumoral sample. The mRNA and protein data are expressed as the median and interquartile range (IQR) of fold change in gene expression level in the gastric tumors normalized to the ACTB gene and relative to levels in the adjacent non-neoplastic control sample.
The relative quantification of gene expression was calculated according to the method of Livak and Schmittgen [71 (link)]. We used a control sample of non-neoplastic gastric mucosa cells MNP01 (Normal gastric mucosa cell Line 01) pooled from 10 healthy patients [16 (link)], which was used as a calibrator for each tumoral sample. The mRNA and protein data are expressed as the median and interquartile range (IQR) of fold change in gene expression level in the gastric tumors normalized to the ACTB gene and relative to levels in the adjacent non-neoplastic control sample.
Free full text: Click here
