Specific quantitect primer assays

Total RNA was isolated from ARPE-19 lysates following the miRNeasy Mini kit (Qiagen, 21,7004). RNA concentration and purity was determined by using the NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA (gDNA) contaminations were eliminated from RNA samples before the reverse transcription (RT) step, carried out on the Gene AMP PCR System 9700 (Applied Biosystems, MA, USA) by using the QuantiTect Reverse Transcription kit (20,5311, Qiagen), according to the protocol “Reverse Transcription with Elimination of Genomic DNA for Quantitative, Real-Time PCR”. The final step for real-time PCR (qPCR) analysis was carried out in triplicate on the CFX96 Real-time System C1000 Touch Thermal Cycler (Biorad). This was performed according to the protocol “Two-Step RT-PCR (Standard Protocol)” by using the QuantiTect SYBR Green PCR Kit (20,4143, Qiagen) and specific QuantiTect Primer Assays (24,9900, Qiagen) for each gene tested (TGF-β1: QT00000728; TGF-βR1: QT00083412; TGF-βR2: QT00014350; AP4S1: QT00197001; LGALS1: QT00064113; ACTA2: QT00088102; and Fn1: QT00038024). Relative quantization of gene expression was performed by using the 2^^−ΔΔCt method [39 (link)] by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (QT00079247) as housekeeping control gene.

Total RNA was extracted from whole intestinal tissue using the peqGOLD Total RNA Kit (peqLab/VWR). cDNA was synthesized by reverse transcription using the SCRIPT cDNA Synthesis Kit (Jena Bioscience) and analyzed by real-time qPCR using SYBRGreen reagent (Roche), the LightCycler 480 (Roche) and specific QuantiTect Primer Assays (Qiagen) or primers ordered from Biomers (for detailed information see Supplementary Table 2). PCR product specificity was verified by performance of a melting curve for each primer set. Experimental values were normalized to levels of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt) or Glyceraldehyde 3-phosphate dehydrogenase (Gapdh). For fold change calculation, the average mean of the relative expression of control mice were set as 1.

Total RNA was extracted from intestinal tissue using the peqGOLD Total RNA Kit or Total peqGOLD Microspin Total RNA Kit for organoids (peqLab/VWR). cDNA was synthesized by reverse transcription using the SCRIPT cDNA Synthesis Kit (Jena Bioscience) and analyzed by real-time qPCR using SYBRGreen reagent (Roche), the LightCycler 480 (Roche) and specific QuantiTect Primer Assays (Qiagen) (for detailed information see Supplementary Table 2). Experimental values were normalized to levels of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt) or Glyceraldehyde 3-phosphate dehydrogenase (Gapdh). For fold change calculation, the average mean of the relative expression of control mice were set as 1.