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).