Script cdna synthesis kit

With 3,000 ng of total RNAs as the template, cDNA samples were prepared in a 20 μl reaction system using the SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). Poly-A tail addition was performed. Briefly, total RNAs samples and oligo (dT) primers were mixed, followed by the addition of RNase-free water to prepare a 10 μl mixture, which was then incubated at 65°C for 10 min. After that, 10 μl of the reaction mix was added to prepare a 20 μl mixture, which was then incubated at 50°C for 60 min, and then 70°C for 10 min. With cDNA samples as template, qPCRs were performed to determine the expression of TRERNA1 and miR-23a. Ct values of TRERNA1 and miR-23a were normalized to the internal controls 18S rRNA and U6.

Immediately upon isolation, DRGs were transferred to TRIzol (Thermo Fisher Scientific) and homogenized using a TissueLyser (Qiagen, Hilden, Germany). Subsequently, RNA was isolated according to manufacturer’s protocol (TRIzol, Thermo Fisher Scientific). The quality of RNA was assessed using a Nanodrop ND1000 Spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized from 500 ng to 1 µg RNA using oligo-dT primer and SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). Quantitative real-time PCR was performed for 40 cycles at an annealing temperature of 60 °C using SensiFast Sybr No-ROX Kit (Bioline, London, UK) in a CFX Connect qPCR System (Bio-Rad Laboratories, Hercules, CA, USA). Primer sequences are listed in supplementary table S1. The detected quantification cycles (C_q) were normalized to C_q values of the housekeeping gene hypoxanthin-guanin-phosphoribosyltransferase (HPRT) using the 2^−ΔΔCT method^{63 (link)}. The amplified DNA products were separated by agarose gel electrophoresis on a 2% agarose gel supplemented with Midori Green Advanced (Nippon Genetics Europe, Dueren, Germany) and visualized using a Gel Doc XR + Gel Documentation System (Biorad, Hercules, CA, USA).

Pooled total RNA (1.0 μg) from 5 plants, from two independent experiments, was retro-transcribed into cDNA according to the manufacturer's indications using the SCRIPT cDNA Synthesis Kit (Jena Bioscience www.jenabioscience.com). RT-qPCR was performed in 96-well plates with the Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific), using SYBR Green Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, www.thermofisher.com). Two independent experiments were analyzed with three technical replicates each. RT-qPCR conditions were as follows: an initial 95°C denaturation step for 15 min followed by denaturation for 15 s at 95°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C for 45 cycles. Gene expression values were normalized using the mean expression of two genes: AT4G26410 and AT1G72150 previously described as stable reference genes (Serrano and Guzmán, 2004 (link); Czechowski et al., 2005 (link)). Normalized gene expression was determined using the comparative 2^−ΔΔCT method previously described (Schmittgen and Livak, 2008 (link)). Primers for ACS6, PR4, and PDF1.2 gene expression were previously described (Hael-Conrad et al., 2015 (link)).

Synthesis of highly structured and long cDNA fragments was performed using SCRIPT cDNA Synthesis Kit (Jena Bioscience, GmbH, Germany) according to the manufacturer’s instruction. Gradient PCR was carried out using the primers pairs, to optimize the annealing temperature and amplification of targeted fragments using MiniAmp Plus Thermal Cycler (Applied Biosystems). The PCR process was performed in a total volume of 50μl comprising 10μl of 5x Red Load Taq Master Mix, 5ul each of 10μM forward and reverse primers, 3ul of cDNA template and PCR grade water to make up the volume. PCR cycling conditions: 1 cycle of 94°C for 2 minutes; 30 cycles of 94°C for 30 secs, 53–64°C for 30secs, 72°C for 2 minutes and 1 cycle of 72°C for 2 minutes were used. The amplicons were analysed by 1.8% agarose gel electrophoresis.

The peqGOLD RNA Kit (732-2868, Peqlab, Erlangen, Germany) was used to extract total RNA from tissues or cultured cells according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using the SCRIPT cDNA Synthesis Kit (PCR-5112, Jena Bioscience, Jena, Germany). Quantitative real-time PCR was performed with specific QuantiTect Primer assays (Qiagen, Hilden, Germany). HPRT or ACTB were used for normalization. Experiments were performed at least two times and representative data are shown.

Huh7 cells were infected with CHIKV and treated with inhibitory compounds as described above. At 6 hpi, total RNA was extracted from cells using TRI Reagent Solution (Applied Biosystems) according to the manufacturer’s instructions. Strand-specific qPCR (ssqPCR) was performed according to the protocol described by Plaskon and colleagues [26 (link)]. Briefly, 500 ng of RNA were reverse-transcribed with gene specific primers (S1 Table) using the SCRIPT cDNA Synthesis Kit (Jena Bioscience) according to the manufacturer’s protocol. 100ng of strand-specific cDNA was used as template for the quantitative PCR performed with the qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems) with gene specific primers (S1 Table) amplifying a 94 bp region of the CHIKV nsP1 encoding sequence using the following PCR program: 95°C for 2 mins, 40 x (95°C for 5 sec, 60°C for 30 sec), dissociation curve 60°C-95°C as pre-defined by the Mx3005P thermal cycler (Agilent technologies). In vitro transcribed CHIKV ICRES RNA was reverse transcribed and a cDNA dilution series employed as a standard to quantify copy numbers in the respective samples. All experiments were performed in four independent repeats, each consisting of 2 wells/condition. A one-way ANOVA was employed and Dunnett’s multiple comparisons test was performed comparing each sample to the untreated control sample.