Bioscript cdna synthesis kit

Total RNA was extracted from EPCs using TRIzol reagent (Invitrogen). Reverse transcription reactions were performed at 42 °C using BioScript cDNA synthesis kit (Bioline). RT-PCR analyses were carried out using MyTaq HS Mix (Bioline) with the following primer pairs: (5′-GAAGTTCAAATGCCCTTCCA-3′ & 5′-TCGATGTGCTTTAGCCACTG-3′) were used for FGFR1; (5′-CAGGGGTCTCCGAGTATGAA-3′ & 5′-TCTCGGAGGTTGCCTTTAGA-3′) were used for FGFR2; (5′-ACTGTCTGGGTCAAGGATGG-3′ & 5′-GTTCTTCAGCCAGGAGATGG-3′) were used for FGFR3; and (5′-CAAAGACAACGCCTCTGACA-3′ & 5′-TGGATACACTTCCGGGACTC-3′) were used for FGFR4. Quantitative real-time PCR (q-PCR) analyses were performed using SYBR Green PCR master mix (Roche) with the following primer pairs: (5′-CTGCCCAGAAGGGAAGCGTGATGA-3′ & 5′-TGACGGACAAGTACAGGCTGC-3′) were used for CXCR4; (5′-GGTACATCCTCGACGGCATCT-3′ & 5′-GTGCCTCTTTGCTGCTTTCAC-3′) were used for IL-6; (5′-ACTGAGAGTGATTGAGAGTGGAC-3′ & 5′-AACCCTCTGCACCCAGTTTTC-3′) were used for IL-8; (5′-CTACCTCCACCATGCCAAGT-3′ & 5′-GCAGTAGCTGCGCTGATAGA-3′) were used for VEGF-A; and (5′-TGCCGACAGGATGCGAAG-3′ & 5′-CGCTCAGGAGGAGCAATGA-3′) were used for actin. The relative mRNA expression levels were calculated according to the ΔΔCt method and normalized to actin.

Total RNA was isolated from frozen liver and epididymal WAT using peqGOLD TriFast (Peqlab, Sarisbury Green, UK). For epididymal WAT, homogenates were centrifuged and the lipid layer was removed before phase separation. cDNA was synthesised from 1ug RNA using bioscript cDNA synthesis kit (Bioline, London, UK). Per sample the mastermix contained 0.5 μl Oligo (dT)₁₈, 0.5 μl Random Hexamer, 1 μl dNTP (10 mM) mix, 4 μl 5 x RT buffer, 1 μl Ribosafe RNase Inhibitor (10 u/μl), 1 μl Tetro Reverse Transcriptase (200 u/μl), and the required volume of DEPC-treated water to make each sample up to 20 μl. Target genes were amplified by quantitative PCR (qPCR) on the LightCycler-480 (Roche, Burgess Hill, UK), using cDNA (2 µl), nuclease-free water (2.5 μl), combined forward and reverse gene-specific primers (0.5 μl) and GoTaq qPCR master mix (5 μl) (Promega, Southampton, UK). Relative mRNA levels were calculated using the Pfaffl method^{42 (link)} and normalized to a geometric mean of three reference genes; Beta-actin, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Ywhaz for the liver, and Nono, Ywhaz and Beta-actin for epididymal WAT.