Sequence detection system software version 2

Gene expression of TNFα, COX-2, VEGF, HIF1α, ACE2, and TMPRSS2 was conducted as previously reported [60 (link)]. Briefly, after extraction through the TRI Reagent, total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ThermoFischer Scientific, Waltman, Massachusetts, USA). Gene expression was determined by quantitative real-time PCR using TaqMan probes obtained from ThermoFischer Scientific (Waltman, Massachusetts, USA). β-actin was used as the housekeeping gene. The analysis of data was conducted with the Sequence Detection System (SDS) software version 2.3 (ThermoFischer Scientific, Waltman, Massachusetts, USA). A detailed description of the experimental protocol is reported in a previous paper of ours [20 (link)].

Gene expressions of COX-2 and BDNF were measured as previously reported [48 (link)]. Briefly, after extraction through the TRI Reagent, total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific). Gene expression was determined by quantitative real-time PCR using TaqMan probes obtained from Thermo Fischer Scientific. β-actin was used as the housekeeping gene. The analysis of data was conducted with the Sequence Detection System (SDS) software version 2.3 (Thermo Fischer Scientific). A detailed description of the experimental protocol is reported in a previous paper of ours [51 (link)].

Gene expression of TNFα, COX-2, VEGFA, IL-6, IL-8, NFkB, and TIMP1 was conducted as previously reported [35 (link)]. Briefly, after extraction through the TRI Reagent, the total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (ThermoFischer Scientific, Waltman, MA, USA). Gene expression was determined by quantitative real-time PCR using TaqMan probes obtained from ThermoFischer Scientific (Waltman, MA, USA). β-actin was used as the housekeeping gene. The data analysis was conducted with the Sequence Detection System (SDS) software version 2.3 (ThermoFischer Scientific, Waltman, MA, USA). A detailed description of the experimental protocol is reported in a previous paper of ours [35 (link)].

Total RNA was extracted from both prostate and ovary specimens using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocol, and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Waltman, MA, USA). Gene expression of TNF-α, IL-6, CAT, and SOD was determined by quantitative real-time PCR using TaqMan probe-based chemistry, as previously described [14 (link)]. PCR primers and TaqMan probes were purchased from Thermo Fisher Scientific Inc. The elaboration of data was conducted with the Sequence Detection System (SDS) software version 2.3 (Thermo Fischer Scientific). Relative quantification of gene expression was performed by the comparative 2^−∆∆Ct method [37 (link)].

Total RNA was extracted from colon specimens using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocol, and reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (ThermoFischer Scientific, Waltman, MA, USA). Gene expression was determined by real-time quantitative PCR using TaqMan probe-based chemistry. PCR primers and TaqMan probes were purchased from Thermo Fisher Scientific Inc. The Assays-on-Demand Gene Expression Products used for gene expression evaluations in the mouse colon specimens were: Mm00478374_m1 for COX-2 gene, Mm00607939_s1 for β-actin gene. β-actin was used as the housekeeping gene. The elaboration of data was conducted with the Sequence Detection System (SDS) software version 2.3 (ThermoFischer Scientific). Relative quantification of gene expression was performed by the comparative 2^−∆∆Ct method [75 (link)].

Total RNA was extracted from both HCT116 cells and colon specimens using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocol, and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Waltman, MA, USA). Gene expression of COX-2, TNF-α, TRPM8, HIF-1α, and VEGF-A were determined by quantitative real-time PCR using TaqMan probe-based chemistry, as previously described [60 (link)]. PCR primers and TaqMan probes were purchased from Thermo Fisher Scientific Inc. The Assays-on-Demand Gene Expression Products used for gene expression evaluations in the mouse colon specimens were Mm00478374_m1 for COX-2 gene, Mm00443258_m1 for TNF-α gene, Mm00607939_s1 for β-actin gene. The Assays-on-Demand Gene Expression Products used for gene expression evaluations in the HCT116 cells were: Hs01066596_m1 for TRPM8 gene, Hs00153153_m1 for HIF-1α, Hs00900055_m1 for VEGFA, Hs99999903_m1 for β-actin gene. β-actin was used as the house-keeping gene. The elaboration of data was conducted with the Sequence Detection System (SDS) software version 2.3 (Thermo Fischer Scientific). Relative quantification of gene expression was performed by the comparative 2^−ΔΔCt method [61 (link)].