Taqman fast advanced master mix protocol

Quantitative real-time PCR was performed according to the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA, USA) according to the Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). included The following primers were used: The housekeeping genes GAPDH (Mm99999915_g1) and GUSB (Mm00446953_m1) as well as TGFβ (Mm01178820_m1), HGF (Hs04329698_m1), CCL17 (Mm01244826_g1), IFNγ (Hs00989291_m1) and TNFα (HS01113624_g1) (Single Tube TaqMan Gene Expression Assays, Thermo Fisher Scientific, Waltham, MA, USA). The analysis was performed using StepOnePlus™ Software v2.3.
The mean cycle threshold (CT) value was calculated for the housekeeping genes. Relative expression values for the analyzed genes were then calculated as the difference between the mean cycle threshold (CT) of the housekeeping genes and the analyzed gene (delta CT) and depicted as the logarithmic value.

10 ng of small RNA fraction isolated from middle temporal gyrus, middle frontal gyrus and hippocampal samples were used to perform RT reactions using the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The resulting cDNA, diluted 1:10, was used as input for qPCR using specific TaqMan Advanced miRNA Assays and the TaqMan Fast Advanced Master Mix Protocol (Thermo Fisher Scientific). IDs of the TaqMan Advanced miRNA Assays used are listed in Supplementary Table S9. GeNorm and NormFinder tools were used to define the most stable miRNAs to be used as endogenous controls, that resulted to be: miR-423-3p (478327_mir), miR-181b-5p (478583_mir) and miR-191-5p (477952_mir). To normalize the data, the arithmetic Ct mean of the three stable miRNAs was evaluated for each sample; then, the miRNA expression levels were calculated according to the ΔΔCt method, setting the arithmetic ΔCt mean of control group as calibrator. The resulting data were represented as log2(ΔΔCt) and were expressed as the mean ± SD. Two-tailed Student’s T tests were performed to assess the statistical significance of miRNA expression levels differences observed between normal and LOAD samples. A p-value < 0.05 was considered statistically significant.

Approximately 1-cm (in length) samples from distal parts of the colon were disrupted and homogenized with the TissueLyser LT (Qiagen, Hilden, Germany) followed by total RNA extraction according to the manufacturer's instruction using RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany) and chloroform (Sigma-Aldrich, St. Louis, MO). No further treatment with DNase was needed because gDNA Eliminator Solution is included in the kit.
For cDNA synthesis, 5 μg of total RNA was used. Reverse transcription was performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany) using QuantiNova Reverse Transcription kit (Qiagen, Hilden, Germany). Samples were diluted with RNase-free water to obtain a cDNA concentration between 10 pg and 100 ng as required by the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA).
RNA and cDNA purity was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).