2.7. Analysis of gene expression by RT-qPCR
Total RNA was extracted from liver and fat samples using
TRIzol® Reagent (Invitrogen Life Technologies,
Carlsbad, CA, USA), following the manufacturer’s recommendations, and
was reverse transcribed into cDNA using oligo(dT) primers (Promega,
Southampton, UK). Real time quantitative PCR (qPCR) amplification and
detection was carried out on optical-grade 48 well plates in EcoTM Real
time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the
KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems,
Wilmington, MA, USA) with specific primers (Table 1). The mRNA relative
quantitation was calculated using the ΔΔCt method and glyceraldehyde
3-phosphate dehydrogenase (Gapdh ) was used as housekeeping gene.
2.8. Flow
cytometry
The cells from adipose and liver tissues were collected following the
procedure previously described with some
modifications(Anderson, Carrillo-Gálvez, &
Martín, 2015). CD45+CD11b+ and
CD11b+Ly6C+