Metaphor agarose gel

Small RNA libraries were prepared from 1 μg total RNA from subcutaneous adipose tissue by the standard TruSeq small RNA sample preparation protocol (Part # 15004197 Rev. E) following manufacturer’s instructions. Briefly, RNA was ligated to a 3’ adaptor followed by ligation to a 5’ adaptor. RNA was reversely transcribed and amplified using TruSeq indexes to barcode the samples. The libraries were size-selected by 3% MetaPhor agarose gel (Cambrex Bio Science, Rockland) for the small RNA fraction including miRNAs and sequenced on two lanes on an Illumina Genome Analyzer IIx.

Brain microvessels were isolated from adult rat brains as published earlier (Veszelka et al., 2007 (link)). For receptor expression analysis total RNA was isolated from isolated brain microvessels and brain endothelial cells by TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). One microgram of total RNA was transcribed to cDNA by Maxima First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA). Specific oligonucleotide primers were designed for the Mc1r gene (XM_006222790). Primer sets were MC1R_fwd 5′-TGCACCTCTTGCTCATCGTT-3′ and MC1R_rvs 5′-ACCTCCTTGAGTGTCATGCG-3′. The predicted length of PCR product was 160 bps. Primers for β-actin were used as internal controls (NM_031144). Primer sets were ACT_fwd 5′-TACTCTGTGTGGATTGGTGGC-3′ and ACT_rvs 5′-GGTGTAAAACGCAG CTCAGTAA-3′. The predicted length of PCR product was 150 bps. PCR was performed with FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia) in T100 thermal cycler (BioRad, Hercules, CA, USA). After initial heat inactivation (95 °C for 3 min) the following cycling conditions were applied: melting 94 °C for 15 s, annealing 50 °C for 15 s, polimerization 72 °C for 20 s (35 cycles). After a final 5 min extension at 72 °C PCR products were analyzed on 3% MetaPhor agarose gel (Cambrex BioScience, Rockland, ME, USA) and fragments were verified by capillary DNA sequencing.