Blue s green master mix

Total RNA was isolated using the NucleoSpin RNA kit (740955.250, Macherey-Nagel, Düren Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 500 ng total RNA using M-MLV reverse transcriptase (M1705, Promega, Walldorf, Germany) and oligo dT primer. Quantitative Real-Time PCR (qRT-PCR) was performed with a Biozym Blue S’Green master mix (331416XL, Biozym Scientific, Hessisch Oldendorf, Germany) on a BioRad CFX Opus 96 System. Relative expression levels were calculated with the ΔΔct method using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reference gene. The following primer pairs were used: HIF1A 5′-GGATGCTGGTGATTTGGATA-3′ (forward) and 5′-TCATGGTCACATGGATGAGTA-3′ (reverse); HIF2A 5′-CGGAGGTGTTCTATGAGCTGG-3′ (forward) and 5′-AGCTTGTGTGTTCGCAGGAA-3′ (reverse); CA9 5′-CACGTGGTTCACCTCAGCAC-3′ (forward) and 5′-CAGCGATTTCTTCCAAGCG-3′ (reverse); HPRT 5′-CCTGGCGTCGTGATTAGTGA-3′ (forward) and 5′-CGAGCAAGACGTTCAGTCCT-3′ (reverse).

Total RNA was obtained from 30 Co-MO or SB-MO clic5 injected zebrafish embryos at 1dpf using the RNeasy Kit (Qiagen). First strand cDNA synthesis was performed using the cDNA Reliance Select Synthesis Kit (BIO-RAD). qPCR was performed on a Light Cycler 480 (Roche) using the Blue S’Green Master Mix (Biozym). 10 µl reaction volumes were used with following cycle program: 95 °C for 2 min (95 °C for 5 s, 60 °C for 30 s) × 44. actb1 and ef1α were used as normalization control. Technical triplicates of four biological samples were analysed for gene expression. Melt curve analysis was performed. The following primers were used for qPCR analysis: actb1 (forward: 5ʹ-CCTTCCTTCCTGGGTATGG-3ʹ, reverse: 5ʹ-GGTCCTTACGGATGTCCAC-3ʹ); ef1α (forward: 5ʹ-TGCCAACTTCAACGCTCAGGTC-3ʹ, reverse: 5ʹ-TCAGCAAACTTGCAGGCGATG-3ʹ); gli1 (forward: 5ʹ-TCAGACGTCCTCTCGCCTTA-3ʹ, reverse: 5ʹ-AGCTCATGTCTCCGATTGCC-3ʹ); ptc1 (forward: 5ʹ-GGGTCCTGAATGGACTGGTG-3ʹ, reverse: 5ʹ-CCGCTGGAGATACCTCAGGA-3ʹ); axin2 (forward: 5ʹ-ACCCTCGGACACTTCAAGGA-3ʹ, reverse: 5ʹ-GTGCAGTCATCCCAGACCTC-3ʹ); wnt8a (forward: 5ʹ-ATTCGTGGATGCGCTTGAGA-3ʹ, reverse: 5ʹ-TTACAGCCAAACGTCCAGCTT-3ʹ); lef1 (forward: 5ʹ-CAGACATTCCCAATTTCTATCC-3ʹ, reverse: 5ʹ-TGTGATGTGAGAACCAACC-3ʹ).