Beacon designer software

RNA were withdrawn using Nucleospin® RNA II Kit (Macherey-Nagel, France) according to the manufacturer's protocol. One μg of total RNA from each sample was reverse-transcribed using the Promega RT system (Promega, France) (RT at 42°C for 1 h). Forward (F) and reverse (R) primers were designed for each gene using Beacon Designer software (Bio-Rad, France): human EPOR: F = 5′-CCTGACGCTCTCCCTCATCC-3′ and R = 5′-GCCTTCAAACTCGCTCTGTGG-3′; human β-actin: F = 5′-GACAGGATGCAGAAGGAGATTACT-3′ and R = 5′-TGATCCACATCTGCTGGAAGGT-3′. Assays were run in duplicate on the iCycler iQ™ real-time PCR detection system (Bio-Rad, France). The amplification profile was as follows: Hot Goldstar enzyme activation, 95°C for 3 min; PCR 50 cycles at 95°C, 15 sec and 60°C, 1 min. The PCR was done according to the manufacturer's protocol using the PCR™ Core Kit Sybr™ Green I (Eurogentec, France). The results were analysed using a comparative method between the fractional cycle number to reach a fixed threshold and the fractional cycle number of β-actin gene and expressed using the 2^−ΔCt formula.

DNase-free RNA was isolated by using PureLink RNA Mini Isolation Kit (Ambion). cDNA synthesis was performed by using the iScript cDNA Synthesis Kit (Bio-Rad). Human primers were designed by using Beacon Designer software (Bio-Rad). Quantitative PCR was performed on a CFX96 real-time PCR thermocycler (Bio-Rad) using the SYBR-Green PCR Master Mix (Bio-Rad). Quantitative PCR was performed in triplicate. Gene expression levels were normalized to human 29S rRNA and plotted as gene expression level and relative expression levels to a housekeeping gene. Values represent mean value.

qPCR was performed to quantify the expression of SRSF2 in siRNA-mediated knockdown experiments. RNA extraction and cDNA synthesis was performed as indicated above. As control, cDNA synthesis of each sample was also performed in the absence of reverse transcriptase in order to detect genomic DNA contamination. SRSF2 expression levels were quantified using the SYBR green technology in a CFX96 Real-Time PCR Detection System (Bio-Rad). Specific primers for SRSF2 and GAPDH were designed using Beacon designer software (Bio-Rad) and the sequences are the following: SRSF2_qPCR_F: 5’-GCCGCAGCCGATCC-3’; SRSF2_qPCR_R: 5’-ACGAGGACTTGGACTTGG-3’; GAPDH_F: 5’-AAGGTGAAGGTCGGAGTCAA-3’ and GAPDH_R: 5’-AATGAAGGGGTCATTGATGG-3’. GAPDH was used to normalize the results. The relative expression levels were calculated using the equation ΔC_T = C_T(target)−C_{T(normalizer)} for Ct normalization and the difference between ΔC_T test (anti-SRSF2 siRNA-treated cells) and ΔC_T calibrator (anti-luciferase siRNA-treated cells) were used to calculate the expression ratio and compare the expression levels [23 (link)]. The data shown are the mean fold induction ± SD from three independent experiments.

Total RNA was extracted using the innuPREP RNA Mini Kit (Westburg, Leusden, The Netherlands) following manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from RNA samples using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). Primer sequences were designed using the Beacon Designer Software (Biorad, Hercules, CA, USA) and used at a final concentration of 500 nM (Table 1). Gene expression was analyzed using Bio-Rad iCycler (iQ5 Real-Time PCR Detection System, Biorad, Hercules, CA, USA) with SYBR Green supermix (Promega). β-actin was used as housekeeping gene. The 2−∆∆CT method was used to determine the relative changes of gene expression.

RNA was withdrawn using Nucleospin® RNA II Kit (Macherey-Nagel, France) according to the manufacturer’s protocol. One microgram of total RNA from each sample was reverse-transcribed using the Promega RT system (Promega, France) (RT at 42 °C for 1 h). Forward (F) and reverse (R) primers were designed for each gene using Beacon Designer software (Bio-Rad, France) as detailed in Additional file 2: Table S2. Assays were run in duplicate on the iCycler iQ™ realtime PCR detection system (Bio-Rad, France). The amplification profile was as follows: Hot Goldstar enzyme activation, 95 °C for 3 min; PCR 50 cycles at 95 °C, 15 s, and 60 °C, 1 min. The PCR was done according to the manufacturer’s protocol using the PCR™ Core Kit Sybr™ Green I (Eurogentec, France). The results were analyzed using a comparative method between the fractional cycle number to reach a fixed threshold and the fractional cycle number of β-actin gene and expressed using the 2^−ΔCt formula.

The qPCR was used to determine the level of Galnts gene expression in livers from wild-type and Galnt2^−/− mice. DNase-free RNA was isolated using the PureLink® RNA Mini Isolation Kit (Ambion). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad). PCR primers used were published previously [23 (link)] using Beacon Designer software (BioRad). The qPCR was performed on a CFX96 real time PCR thermocycler (Bio-Rad) using the SYBR-Green PCR Master Mix (Bio-Rad). The qPCR was performed in triplicate, and four independent experiments were performed. Gene expression levels were normalized to 29S rRNA and displayed as relative expression levels.