Rnase free dnase

Frozen plant material from second internodes or the stem base (including 5 mm above) of 15–20 cm tall plants (three biological replicates (three plants each) per genotype/treatment) were pulverized with pestle and mortar and RNA was isolated by phenol/chloroform extraction. RNA elution in RNase-free water was followed by treatment with RNase-free DNase (Thermo Fisher Scientific) and reverse transcription (RevertAid First Strand cDNA Synthesis Kit; Thermo Fisher Scientific). cDNA was diluted 1:25 prior to amplification. qRT-PCR was performed using SensiMix™ SYBR® Green (Bioline Reagents Ltd) mastermix and gene-specific primers (listed in Supplementary Table 1) in a Roche Lightcycler480 following the manufacturer’s instructions. Experiments were performed in triplicates with plant material of three plants being pooled for each replicate. Two reference genes (ACT2 and EIF4a) were used to normalize our signal. Error bars: ±standard deviation. Raw amplification data were exported and further analysis and statistical tests were done using Microsoft Excel 2010.

Roots from 9 avocado plants from control, mild-WS and severe-WS were harvested at t₁ and t₂ in plants others than those used in the pathogenicity test. Three biological replicates were used for RNA extraction. Each replicate consisted in a bulk sample from three plants. RNA from ground root tissue was extracted using the CTAB extraction method [113 (link)], a simple and efficient method for isolating RNA from pine trees with slight modification. The chloroform:isoamyl alcohol step was repeated 3–5 times, depending on the stability of the interphase and colour of the sample. RNA quantity and quality were determined based on A260/280 and A260/230 wavelength ratios using a NanoDrop® ND-1000 (Nanodrop Technologies, Inc., Montchanin, USA) spectrophotometer. RNA integrity was confirmed by the appearance of ribosomal RNA bands and lack of degradation products after separation on a 2% agarose gel and Red Safe staining. DNase treatment of RNA was performed by the addition of 1 U RNase-free DNase (Thermo Scientific, Life Technologies Inc., Carlsbad, California, USA), 1 μL 10x reaction buffer with MgCl₂, 1 μg RNA, 0.5 μL of RiboLock RNase Inhibitor (Thermo Scientific Inc., California, USA) and diethylpyrocarbonate-treated water to a final volume of 10 μL. The mixture was incubated at 37 °C for 45 min followed by the addition 1 μL of 50 mM EDTA and incubation at 65 °C for 10 min.

RNX-Plus reagent (SinaClon, Iran) was used to isolate total RNA, according to the
manufacturer’s protocol. RNA was quantified, and the concentration and purity were
measured based on absorption rate of 260/280 nm using a Nanodrop spectrophotometer
(Nanodrop 2000, Thermo Scientific, USA). Total RNA samples were treated with RNase-free
DNase (Thermo Scientific, USA) before quantitative reverse transcription polymerase chain
reaction (qRT-PCR). A RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA)
was used to synthesize complementary DNA (cDNA) from total RNA. A PCR test was applied
using tst specific primers on cDNA to confirm tst gene
expression after lipofection. Primer sequences were as follows:
tst-
Sense: 5´-GCACAAACGACAACATTAAGGACC-3ˊ
Antisense: 5´-TTGTCCGCTTTGTGTTGAGGTC-3ˊ.

Avocado roots from control, mild-WS, and severe-WS plants were harvested at t₁ and t₂. Three biological replicates per timepoint, in which each biological replicate consisted of three plants (n = 9), were used for RNA extraction in each experimental group. RNA from ground root tissue was extracted using the CTAB extraction method [161 (link)] with modification described by Zumaquero et al. [21 (link)]. RNA parameters and integrity were checked using a NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA) spectrophotometer based on the A260/280 and A260/230 wavelength ratios and running samples on a 2% agarose gel. RNA samples were treated with a DNase treatment with 1 U of RNase-free DNase (Thermo Scientific, Life Technologies Inc., Carlsbad, CA, USA), 1 μL of 10× reaction buffer with MgCl₂, 1 μg of RNA, 0.5 μL of RiboLock RNase Inhibitor (Thermo Scientific Inc., CA, USA), and diethylpyrocarbonate-treated water to a final volume of 10 μL in all RNA samples. The mixture was incubated according to the manufacturer’s instructions at 37 °C for 45 min followed by the addition of 1 μL of 50 mM EDTA and incubation at 65 °C for 10 min according to the manufacturer’s instructions.

CWR22Rv1 cells were infected with AdTR3 or AdCtrl, maintained for 24 h, and cross-linked with 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The fixed cells were then subjected to cross-linked RNA-immunoprecipitation (CLIP) analysis as previously described [17 (link)] with minor modifications. The protein-bound primary RNA fragments were enriched through immunoprecipitation with anti-hnRNP A2B1 antibodies (Table S2). DNA contaminants were removed by treating with RNase-free DNase (ThermoFisher Scientific, Rockford, IL, USA) at 37 °C for 30 min, following which, RNA fragments were obtained using chloroform extraction, and cDNA was synthesized using random hexamers (Enzynomics) and M-MLV Reverse transcriptase kit (Promega, Madison, WI, USA). Enrichment of AR pre-RNA fragments was analyzed via RT-PCR using the CLIP primers listed in Table S1.

A semi-quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of NOX1, p47^phox and HIF-1α messenger RNAs (mRNAs) in 22Rv1 cells was performed using gene-specific primers. Total RNA was extracted from DMSO or GPE-treated 22Rv1 cells with Tri reagent (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instructions. Total RNA was treated with RNase-free DNase (Thermo Scientific, Rockford, IL) and the integrity of RNA was determined by running on agarose gel prior to further downstream application. The RT-PCR was carried out using one-step RT-PCR kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and total RNA (200 ng) was taken as template for each reaction. β-actin gene was taken as endogenous internal standard. NOX1 oligonucleotide sequences for PCR primers were 5′-GGTTGTTTGGTTAGGGCTGA-3′ and 5′-CTGGAGAGAATGGAGGCAAG-3′. p47^phox oligonucleotide sequences for PCR primers were 5′-CCCGATACCCAGTTTCAGTG-3′ and 5′-GGAAGGTCTCCTTGAGGGTC-3′. HIF-1α oligonucleotide sequences for PCR primers were 5′-CAGAGCAGGAAAAGGAGTCA-3′ and 5′-AGTAGCTGCATGATCGTCTG-3′. β-actin oligonucleotide sequences for PCR primers were 5′-TCCTTAATGTCACGCACGATTT-3′ and 5′-GAGCGCGGCTACAGCTT-3′.