Sensimix sybr fluorescein kit

Total RNA was extracted using a Qiagen RNeasy kit with DNase treatment according to the manufacturer’s specifications. Reverse transcription (RT) was carried out with the ImProm II RT kit (Promega) using oligodT as primer. Random hexamer was used as primer for NS5 experiments. Real-time PCR analysis was carried out using the SensiMix SYBR & Fluorescein kit (BioLine) and the following primers; mGAPDH 5′-TGCCCAGAACATCATCCCTG-3′ and 5′-ATCCACGACGGACACATTGG-3′, mIL-6 5′-GACTTCACAGAGGATACCACTCC-3′ and 5′-TTCTGCAAGTGCATCATCGGT-3′,mIFNβ 5′-GGAGATGACGGAGAAGATGC-3′ and 5′-CCCAGTGCTGGAGAAATTGT-3′, Kunjin NS5 5′-GAGTCCAAGAAGTCAGAGGGTACA-3′ and 5′-CCACTCTTCATGGTGACAATGTTCC-3′. Reactions were set up in 96-well PCR plates (Genesee). Amplifications were carried out for 50 cycles, followed by a melt curve analysis of resulting products to confirm the specificity of the reactions. To construct standard curves, total RNA was isolated from the cells, and 300- to 600-bp fragments of the gene of interest encompassing the real-time PCR primer binding sites, were amplified by RT-PCR using the appropriate primer sets. PCR fragments were gel purified and quantified, and the copy number was calculated. Serial 10-fold dilutions were prepared for to create standard curves for real-time qPCR. All data are expressed as the ratio of copy numbers of target gene per 10³ or 10⁴copies of GAPDH as indicated.

RNA was purified from E12.5 and E13.5 palatal tissue or mandibles from wild type and mutant samples using Trizol reagents (Invitrogen). Following RNA extraction, cDNA was synthesized using iScript^™ cDNA Synthesis Kit (BIO RAD). Quantitative PCR amplifications were performed in a StepOnePlus real-time PCR machine (Applied Biosystems) using the SensiMix^™ SYBR & Fluorescein Kit (Bioline). For each gene, the PCR reaction was carried out in triplicates and the relative levels of mRNAs were normalized to that of HPRT using the normalized expression method. Student’s t-test was used to analyze the significance of difference and a P-value less than 0.05 was considered statistically significant.

To determine the tissue specific expression of zfOatp1d1 and zfOatp1f1-4, cDNA was synthesized as described above from adult zebrafish tissues and used as a template for semi-quantitative real time PCR. A 1:5 dilution of the cDNA was used. 18S-rRNA served as normalization control due to its equal expression in all tissues (data not shown). Gene specific primers were designed for Oatp1d1 (forward 5′-CACAATCCTCCTGCCAGCAAA-3′, reverse 5′-CCCTATGAAACCACTGACTTGT-3′) and Oatp1f (forward 5′-GGTATAGGAACACTGCTAATGG-3′, reverse 5′-CAGAGGGATAATACTGGCTTC-3′), whereby primer pairs for zfOatp1f1-4 were 100 % identical for all individual sequences. SensiMix™ SYBR® & Fluorescein Kit (Bioline, London, UK) was used according to the manufacturer's instructions. A melting curve analysis of the products was performed at the end of the PCR reaction to confirm the detection of a single PCR product. Standard curves for primers used were carried out in n=2-4 independent replicates, whereby each replicate consisted of technical triplicates. The resulting efficiency of amplification was 104±4 % for 18s-rRNA (n=4), 98±1 % for zfOatp1d1 (n=3) and 103±0 % for zfOatp1f (n=2) (data not shown). Data were analyzed according to the Δct method.

RNA was obtained using the Trizol method (Invitrogen). The RNA pellet was dissolved in DEPC-treated water (Sigma-Aldrich) and quantitated using absorbance at 260nm on a Nanodrop 200 (Thermo Fisher). 2μg of RNA was used in the first strand synthesis reaction. The High Capacity RNA-to-cDNA Kit (Applied Biosystems) was used according to manufacturer recommendations. The 20μL reaction volume was diluted with 380μL of dH₂O. 5μL of the cDNAs were combined with SYBR Green (SensiMix SYBR & Fluorescein Kit, Bioline), and with 250nM each of forward and reverse primers. The real-time PCRs were run on a CFX96 Real-Time PCR Detection System (Bio-Rad) using the following thermocycling conditions: 1) 95°C 10 minutes, 1× 2) 95°C 15 seconds, 57°C 15 seconds, 72°C 15 seconds, 40×. A melt curve analysis was included at the end of each run. No-transcript controls (NTC) were used to assess for genomic DNA contamination. The data was analyzed using the ΔΔC_t method. Primer sequences can be found in Supplementary Table 2.