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 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 (RNA integrity
number, varies between 1 – 10 with 10 when there is no degradation)
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
(average RIN 8.0±1.21 , see also data on Dryad ). All the results
presented in the Result section therefore do not include data on the
liver samples.