Sybr green 1

Total RNA from HeLa, HL60, U937, and THP-1 cell lines and from different subpopulations of PBMCs (pDC, NK, MO, CD40+, and B) was extracted using the TRIzol reagent (Invitrogen). For experiments with interferon stimulation, HL60 and U937 cells were either mock treated or incubated with 250 units/mL of IFN-α2a (Miltenyi Biotec) and harvested after 48 h. The RNA was converted to cDNA using an oligo(dT)18 primer and reverse transcriptase (Invitrogen). Reverse transcriptase quantitative PCR (RT-qPCR) reactions were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using SYBR Green I (Eurogentec). All reactions were performed in triplicate and experiments were repeated at least twice. Relative amount of mRNA was calculated using the comparative Ct method (Applied Biosystems): 2^−ΔΔCt = 2^{−[(Ct_target−Ct_housekeeping)  sample−(Ct_target−Ct_housekeeping)  control]}, where Ct is the threshold cycle and HPRT or β-microglobulins are the housekeeping genes.

mRNA isolation was performed with Trizol (Invitrogen), according to standard procedure. QRT-PCR was carried out with SYBR® Green I (Eurogentec, Seraing, Belgium) and Multiscribe Reverse Transcriptase (Applied Biosystems) and monitored by an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Detection of RPL14 gene was used to normalize the results. Primer sequences for each cDNA were designed using either Primer Express Software (Applied Biosystems) or qPrimer depot (http://primerdepot.nci.nih.gov) and are available upon request.

The total root system of 7 week-old plants (n = 18) was harvested every 4 h during the 22-h LD and at the same times in control 8-h SD. Roots were stored at −80 °C until used. Tissues were ground in liquid nitrogen and RNA was extracted with TRizol according to manufacturer’s instructions (www.lifetechnologies.com). RNA samples were treated with DNase (0.2 U DNase μg⁻¹). We synthesized first-strand cDNA from 1.5 μg RNA using MMLV reverse transcriptase and oligo(dT)₁₅ according to manufacturer’s instructions (http://www.promega.com). Quantitative PCR (qPCR) reactions were performed in triplicates using SYBR-Green I (http://www.eurogentec.com) in 96-well plates with an iCycler IQ5 (http://www.bio-rad.com). We extracted quantification cycle (Cq) values using the instrument software and imported the data in qbase^PLUS 2.0 (http://www.biogazelle.com). A GeNorm analysis62 (link) was performed in a preliminary experiment to identify suitable reference genes. We selected ACTIN2 (ACT2) and TUBULIN2 (TUB2) (geNorm M value <0.2). The computed geometric mean of their Cq values was used to calculate the normalization factor, as in Vandesompele et al.62 (link). Primers are listed in Supplementary Table 7.

mRNA was extracted using the NucleoSpin RNA II kit (Bioke) using iScript cDNA Synthesis Kit (BioRad). Both methods were performed according to the manufacturers’ instructions. Quantitative PCR was performed on the CFX96 (Biorad). The abundance of the genes of interest were detected with SYBR^® Green I (Eurogentec). Values for each gene were normalized to 18S expression levels. Primer sequences are listed in the S1 Table.

The real-time RT-PCR procedure was conducted to assess the efficiency of the retroviral transfection and to validate the microarray experiment. Total RNA was isolated using TRIzol reagent (Life Technologies). The cDNA synthesis was performed using 10 μg of total RNA at a volume of 100 μl using ImProm RT-II™ reverse transcriptase (Promega). Reverse transcription was carried out under the following conditions: incubation at 25°C for 5 min and 42°C for 60 min and heating at 70°C for 15 min. cDN A samples were diluted with sterile deionized water to a total volume of 150 μl, and 2 μl was added to the PCR reaction. Real-time RT-PCR was performed using Light Cycler 480 (Roche). PCR products were detected using SYBR^® Green I and qPCR Core kit for SYBR^® Green I (Eurogentec). All reactions were performed in duplicate. The relative expression levels of the WWOX, BIRC5 and ID3 genes were assessed. The expression levels of the investigated genes were normalized to 3 reference housekeeping genes (RPS17, H3F3A, RPLP0). The relative gene expression was calculated based on the Pfaffl method (11 (link)). Universal Human Reference RNA (Stratagene) was used as a calibrator. The primer sequences, PCR reaction conditions and lengths of the obtained products are available upon request.

cDNA was synthesized using 1 μg RNA and Superscript^™IIReverse Transcriptase (Life Technologies, Breda, The Netherlands). SYBR Green I (Eurogentec, Maastricht, The Netherlands) was used as detector dye for qRT-PCR of the Abca3, Abcc3, Cs, Fmo3, Elolv3, Cbr1, Cs, G6pc, Mup2, Nudt7, Prodh, Slc22a7, Stat5b, Thrsp (Spot14), Dio1, Dio3, Serca1, Serca2, Ucp3, Slc2a4, Glut4, Hcn2, Myh6 (MHC-α), Myh7 (MHC-β), Mct8, Mct10, Ntcp, Lat1, Lat2, Oatp1a1, Oatp1a4, Oatp1b2, Thra and Thrb. genes. Primer sequences are provided in S1 Table. The housekeeping genes B2M, RPS9 and tubulin were used for normalization.