RNA was isolated from mouse tissue after homogenization in DNA/RNA Shield Stabilization Solution (Zymo, Irvine, CA) using Quick-RNA Miniprep Plus kit (Zymo) according to the manufacturer’s instructions. The resulting RNA was checked for quality using a Nanodrop. cDNA was synthesized by reverse transcription-PCR using SuperScript II Reverse Transcriptase Kit and oligo dT primers (Invitrogen, Carlsbad, CA). Real time PCR was carried out as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min with SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA). Gene expression was evaluated using the ΔΔCT method normalizing to the housekeeping gene m36b4 (F: 5′-ACCTCCTTCTTCTTCCAGGCTTT-3′, R: 5′-CCCACCTTGTCTCCAGTCTTT-3′) as described in (6 (link)). Genes evaluated are as follows: BAATF – 5′-TGTGATGAATAGCCCCTACCA-3′; BAATR – 5′-AGGACTGACGACTATGTCTTGTA-3′ (23 (link)), ACNAT1F - 5′-GAGGCAGCAACTGTGGTGACT-3'; ACNAT2R - 5′-TGAGACTGTATGTTTTCCTTGCTCTAC-3', ACNAT2F - 5′-AAGCGGGAACAGATTCAAGAAG-3′; ACNAT2R - 5′-ACGAAATTCAACTAGACCCCCA-3′ (24 ).
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