Trizol reagent

Peripheral blood and anal mucosa samples were obtained from each subject; the whole blood was used to obtain plasma and serum, to extract DNA, as well as for peripheral blood mononuclear cell (PBMC) isolation. The anal mucosa sample was used for cytology analysis and mRNA extraction and is collected using an optimized protocol as follows: the cytobrush was inserted 5 cm beyond the anal verge, close to the anal wall, and rotated slowly while being withdrawn to capture cells. Then, the sample was spread onto slides for cytology analysis and the cytobrush’s head was cut and put into a vial with RNAlater reagent. A second cytobrush was inserted to obtain more mucosal samples, and this head was put into an RNAlater reagent (Invitrogen). Samples were preserved at 4°C. Then, the cytobrush’s heads were removed and the cell pellet was obtained by centrifugation. TRIzol Reagent (Zymo) was added to lyse the cells. The samples were preserved at −80°C until RNA extraction. The anal sampling and cytology analysis was done by qualified staff from the reference lab “Laboratorio Clínico VID.”

Total RNA was extracted from exosomes and cell lysates using TRIzol reagent as per the manufacturer’s protocol (ZYMO RESEARCH). RNA was eluted with 25 μL of RNAse-free water. RT-qPCR was performed using an Absolute Blue QPCR SYBR Green Low ROX mix (Thermo Scientific) on an Applied Biosystems’ 7500 real-time PCR system. The Rn value (normalized reporter value) was the fluorescent signal from SYBR Green normalized to the signal of the passive reference dye for a given reaction. No-template and no-RT reactions were performed as negative controls. All assays were performed in 3 separate RTs followed by triplicate qPCR, and the results are shown as the average fold change relative to GAPDH which served as an internal control. Primers for RT-qPCR were:

Total RNA was extracted from endometrium stem cells and 1 × 10⁶ mononuclear cells using TRIzol^® reagent (Zymo Research, Irvine, CA, USA) and Quick-DNA/RNA™ Miniprep Kit (Zymo research, Irvine, CA, USA) respectively according to manufacturer’s instructions. cDNA was synthesized with LunaScript^® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA) following manufacturers recommendations. RT-qPCR was performed using Luna^® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) and Rotor-Gene 6000 Real-time Analyzer (Corbett Life Science, QIAGEN, Hilden, Germany) with experimental conditions as follows: 95 °C 1 min, 95 °C 15 s, 60 °C 30 s. The list of primers and their sequences used in gene expression analysis is presented in Table 3. The acquired data from RT-qPCR was further analysed, each sample’s mRNA levels were normalised to GAPDH gene expression and relative gene expression was calculated using ∆∆Ct method.

Bone marrow plasma cells (CD138+), non-plasma cells (CD138−), dendritic cells (CD115+ Ly6C− CD11c+), neutrophils (CD115− Ly6C− CD11b+ Ly6G+), macrophages (CD115− CD11c− F4/80+) and monocytes (CD115+ Ly6C+ CD11c−) were sorted using a BD FACS ARIA III. RNA was isolated using Trizol reagent according to the manufacturer's instructions (Zymo Research). Reverse transcription reaction of total RNA was performed using a QuantiNova Reverse Transcription Kit (Qiagen), including the procedure for removal of contaminating genomic DNA, according to manufacturer's instructions. Quantitative PCR was done using QuantiNova SYBR Green PCR Kit (Qiagen) iQSyber green (Biorad) on a CFX96 real-time PCR system (Biorad) using the specific following primers (Metabion): IL-10 forward 5′-GCGCTGTCATCGATTTCTCC-3′ and reverse 5′-GGCCTTGTAGACACCTTGGTC-3′; IL-10RA forward 5′- GAGCCTAGAATTCATTGCATACG-3′ and reverse 5′-GTACTGTTTGAGGGCCACTT-3′; actin forward 5′-GCACCACACCTTCTACAATGAG-3′ and reverse 5′-AAATAGCACAGCCTGGATAGCAAC-3′ (used as internal control for all samples). Real-time RT-PCR data were analyzed using CFX Manager Software 3.1 (Bio-Rad).

The cochlea from treated and non‐treated five male mice (12 m) was dissected. Total RNA from each cochlear tissue was extracted using Trizol Reagent (Zymo Research, #R2050‐1‐50) and Direct‐zol RNA Miniprep (Zymo Research, #R2050) as described previously (Vikhe Patil et al., 2015 (link)). Next, one microgram of isolated RNA was reverse transcribed using the iScript™ cDNA Synthesis Kit (BioRad). Using the DyNAmo HS SYBR green qPCR kit (F‐410L, ThermoFisher Scientific) with the CFX connect real‐time PCR detection system (Bio‐Rad), qPCR was performed. Primer sequences are listed in Table S1. Experimental values were normalized to values for GAPDH. The same protocol was followed for RNA isolation from cochlear cells except those steps to break the cochlear bone were skipped.