2.2 RNA extraction
RNA extractions and the following laboratory procedures described below were carried out by a private company (Admera Health). The same extraction protocol was used for each of the different tissues and generally followed manufacturer instructions for QIazol (Qiagen) extraction. Briefly, up to 10 mg of tissue was mechanically homogenized in 500 µl of QIazol. After homogenization, QIazol was added to reach 1ml and then 200 µl of chloroform were added and mixed. For blood samples, they were centrifuged at 2000 g for 5 minutes, the supernatant discarded and 1ml of QIazol (Qiagen) added to the tube. Tubes were then left at room temperature for 5 minutes and vortexed a few times to ensure homogenization of the sample. 200 µl of chloroform was added and mixed. All samples (blood or other tissues, all containing 1ml of QIazol and 200 µl of chloroform) were then incubated at room temperature for 3-5 minutes and centrifuged at 4 °C, 12,000 g for 15 minutes. The upper aqueous RNA containing phase was transferred to a new tube. An equal volume of 70% ethanol was added and mixed. The mixture was loaded into a RNeasy mini prep column (Qiagen RNeasy Mini Plus Kit) and RNA eluted following the manufacturer’s protocol.
The quality of RNA was determined by Qubit HS RNA assay (ThermoFisher), and the integrity of RNA was evaluated based on RIN acquired via capillary gel electrophoresis performed using Bioanalyzer 2100 (Agilent Technologies). ANOVA was run in R using the F-test to compare RIN numbers among the samples belonging to the different groups (see below) and to compare the RIN numbers among samples belonging to different tissues in each group. These analyses were run without data from the liver for which most samples showed signs of degradation (see Results and Supporting Information Table S1).