4.2. Polymerase Chain Reaction and qPCR- SYBR Green

Total RNA was isolated from cells using Trizol^TM (Thermo Fisher Scientific) as per the manufacturer’s instructions. A quantity of 1 µg of RNA (260/230 > 1.8 and 260/290 > 1.8) was used to synthesize cDNA using ReadyScript^TM synthesis mix (Sigma RDRT). In addition, 1 µL of cDNA was amplified using JumpStart REDTaq ReadyMix Reaction Mix (P0982); products were run on a 1% agarose gel and imaged in ProteinSimple FluorChem M^TM. Densitometric analysis was performed using AlphaView Software. Primers used included Sortilin exon 17 S 5′-CAAATGCCAAGGTGGGATGAA-3′ and sortilin exon 19 AS 5′-TGACAAGCATCAGTCCCACGAT-3′ to see splice variants, sortilin exon 17b S 5′-AATCCAGCTCTGCCTCCTCT-3′, Glut4 S 5′-GGTGTGGTCAATACGGTCTTCAC-3′ and AS 5′-AGCAGAGCCACGGTCATCAAGA-3′, and β-actin S 5′ GTGGGCCGCTCTAGGCACCAA-3′ and AS 5′-CTCTTTGATGTCACGCACGATTTC-3′. For qPCR absolute quantification, amplification was performed on the Viia 7 (ABI) and primer concentrations were optimized to give a single melt curve. Plate set up included a standard series, no template control and no reserve transcriptase control. A standard curve was generated and used to calculate absolute quantities of target expression using primers above. Samples were normalized to β-actin for absolute quantification.