Rq dnase

Total RNA was extracted from A. castellanii infected with C. gattii WT and Δzip1 after 3 and 24 h of interaction using Trizol^® reagent (Invitrogen) according to the manufacturer’s recommendations. RNA integrity was assessed by electrophoresis on a 1% agarose gel and RNA concentration was measured by spectrophotometry (NanoDrop 2000 spectrophotometer, Thermo Scientific). The samples were treated with RQ DNase (Promega) to purify RNA. Reverse transcription and cDNA synthesis were performed with ImProm-II Reverse transcriptase (Promega) using oligo-dT. The relative expression of genes identified as zinc transporters by the conserved domain (PF02535 and PF01545) of their coding products in A. castellanii were determined by qRT-PCR (StepOne Real-Time PCR System) with an initial step of 95°C for 10 min, followed by 50 cycles of 95°C for 15 s, 55°C for 15 s, and 60°C for 60 s. All experiments were performed in biological triplicate and each cDNA sample was also analyzed in technical triplicate for each primer pair. A melting curve analysis was performed at the end of the reaction to confirm the presence of a single PCR product. The results were processed according to the 2^-ΔCt method (Schmittgen and Livak, 2008 (link)) and relative transcript levels were normalized with actin transcript levels. The primers are listed in Supplementary Table S1.

Specific primers were designed using Primer3 software (Elixir, Narva maantee, Estonia) [41 (link)]. Primers for the various genes were designed to amplify a region containing more than one exon, so that genomic DNA would not be amplified. A total of 2.5 µg RNA from each sample was treated with RQ DNase (Promega, Madison, WI, USA) and reverse-transcribed using random hexamer primers (Promega). Real-time PCR was carried out using the SYBR green amplification kit (ABgene, Blenheim Road, Epsom, UK) according to manufacturer’s instructions. Each reaction contained 1 µL cDNA and 1 µM of each primer from the relevant primer pair in a final volume of 10 µL. Quantification of real-time PCR products was carried out by detection of SYBR green fluorescence on a StepOnePlus™ system (Applied Biosystems, Foster City, CA, USA). Dilution series of cDNA were created for each set of and a standard curve was established for each gene. Triplicate of cDNA were used and each reaction was subjected to melting-point analysis to confirm single amplified products. At least two biological repeats were carried out for each gene. Transcript levels were estimated using a standard curve for each gene, and these levels were normalized against the amount of OeACTIN transcript level in each sample. The sequences of the primers used are listed in the Table S1.

Total RNA was extracted according to Jaakola et al. (2001) (link). The RNA concentration was determined in a Nanodrop ND-1000 spectrophotometer, and its integrity was checked by running 1 μl in a 1% (w/v) agarose gel stained with Bromophenol Blue. Total RNA was digested with RQ-DNase (Promega). Complementary DNA was synthesized, using Oligo-dT primers, using a VERSO cDNA kit (Thermo Scientific).

Fungal cells were pelleted by centrifugation and snap-frozen in liquid nitrogen. The cells were homogenized by bead beating in the presence of glass beads (425 µm to 600 µm) and TRIzol (Ambion), and RNA was extracted following the manufacturer’s instructions. Residual DNA in RNA samples was removed by treatment with RQ DNase (Promega). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega). Expression of PHO genes (listed in Table 1) was quantified by quantitative PCR (qPCR) on a Rotor-Gene 6000 (Corbett Research) using Platinum SYBR green qPCR SuperMix-UDG (Life Technologies, Inc.). Primers used for qPCR are listed in Table S1 in the supplemental material. The expression of each gene was quantified using the 2^−ΔΔCT method, where actin (ACT1 [CNAG_00483]) served as a reference (housekeeping) gene.

RNA extractions were performed on three biological replicates of T1 generation transgenic tobaccos expanding leaves using the TRIzol^® (Invitrogen, USA), while RNA extractions for Kalanchoe daigremontiana were performed⁴⁴ . All samples were treated using RQ DNAse (Promega, USA) and were ethanol precipitated. cDNA synthesis was performed using GoScript^TM Reverse Transcription Systemfor RT-PCR (Promega, USA) with 2 µg RNA and using the oligo (dT) primer. RT-qPCR was performed using the TransStart® Tip Green qPCR SuperMix (TransGenBiotech, China), following the manufacturer instructions and an ABI Step One PCR instrument (Applied Biosystems, USA) was used. 200 ng cDNA was used for each reaction. The results were calculated using the 2^−∆∆CT method^{45 (link)}. The KdActin were used as housekeeping gene and the primers used in RT-qPCR were listed in Table S1.

Total RNAs from A. thaliana WT and nuc1.2 plant mutants were extracted using Trizol reagent (Invitrogen), according to manufacturer's instructions. Then, all samples were then treated with RQ-DNase (Promega) to eliminate contaminant genomic DNA. Primer extension analysis to detect TIS and P sites was done using 5–10 μg of RNAs and specific 5′end labeled primers, as previously described (Sáez-Vasquez et al., 2004b (link); Pontvianne et al., 2007 (link)). Products of the reaction were analyzed on 8% polyacrylamide/ 7 M urea sequencing gel.