Gene expression of 11-βHSD1, 11-βHSD2 and cytokines determined by
RT-PCR in lung and liver
The right lungs of three mice per time point were rapidly obtained and
stored in 1.5-ml cryotubes, immediately frozen in liquid nitrogen and
maintained at −80◦C until processing. For homogenization, lungs were
slowly defrosted, and zirconium flint beads were added to each tube and
lung tissue was homogenized in the FastPrepR-24 (MP Biomedicals). RNA
extraction was performed with the RNeasyR Mini kit (Qiagen) following
the manufacturer’s instructions.
Total RNA was quantified by spectrophotometry (A260/280), 100 ng of RNA
from each lung was used for the production of cDNA by
retro-transcription, following the indications of the Omniscript kit
(Qiagen), and later an endpoint PCR was run to amplify the constitutive
housekeeping gene RPLP0 (Ribosomal protein large P0, Gen ID:11837,
GenBank, NCBI) and its integrity was analyzed by running at 2% agarose
gel stained with SYBR green.
Complementary DNA (cDNA) obtained from each sample was analyzed by
real-time PCR (qPCR) using the Real-Time PCR system 7500 (Applied
Biosystems) and the Quantitech SYBR Green Mastermix kit (Qiagen) with
specific primers designed with the first BLAST (ncbi.nlm.nih.gov) for
11-βHSD1, 11-βHSD2, IFN-γ and TNF-α.
Regarding to relative quantification of 11-βHSD1, 11-βHSD2, the Ct
values were determined by 7500 real-time PCR system (Applied Biosystems,
Foster City, CA, USA), and the fold change of gene expression was
calculated by the 2−(△△Ct) method (29). For absolute quantification, the
number of copies of each target gene was normalized to 1 million
amplicons of mRNA of the housekeeping gene RPLP0, including the standard
curves and a negative control to IFN-γ, TNF-α and 11-βHSD1, 11-βHSD2 in
liver. Cycling conditions used were as follows: initial denaturation at
95◦C for 15min, followed by 40 cycles at 95◦C for 20 s, 60◦C for 20 s,
and 72◦C for 34 s.