Hybond nx nylon membrane

Relative amounts of high-molecular-weight (HMW) and low-molecular-weight (LMW) RNA were assayed in the agroinfiltrated leaf areas by northern analysis. Total RNA was isolated from frozen leaf material using Trizol (Invitrogen, Carlsbad, CA, USA). HMW and LMW RNAs were separated into different fractions using LiCl precipitation, and an antisense RNA probe for detection of gfp mRNA and siRNA was prepared and radioactively labeled with [α-³²P]UTP (PerkinElmer) [55 (link)]. LMW RNA (1.5–2 µg) was separated using denaturing urea polyacrylamide gel (15%) electrophoresis, electroblotted onto Hybond-NX nylon membrane (GE Healthcare), and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [59 (link)]. HMW RNA (3–5 µg) was analyzed by agarose-formaldehyde gel electrophoresis, transferred onto Hybond-NX nylon membranes (GE Healthcare) by capillary blotting, and crosslinked with UV light [49 ]. Hybridization, washing, and autoradiography were done as described [55 (link)]. Experiments were repeated two times.
Radioactive signals were detected using the IP screen (Kodak) and PhosphorImager (FLA-5001, Fuji, Tokyo, Japan). Relative intensities (signals) were quantified using Bio-Rad Quantity One v4.6.9 software (www.Bio-Rad.com). Signals were normalized to RNA loading amount.

Total RNA was isolated from P. capsici–infected and control Col-0 leaf samples using the TRIzol reagent (Invitrogen, United States). The quality and concentration of total RNA were determined by denaturing gel electrophoresis and NanDrop ND 100x. Northern blot analysis was conducted as described previously (Qiao et al., 2015 (link)). Briefly, approximately 20 μg of the total RNA of each sample was analyzed on a denaturing 19% polyacrylamide gel and transferred to Hybond-NX nylon membranes (GE Healthcare, Madison, WI, United States), which were subsequently crosslinked using a Stratagene UV Crosslinker. DNA oligonucleotides complementary to different sequences of miRNAs were synthesized and labeled with biotin (TaKaRa). The membranes were prehybridized with PerfectHyb (Sigma) hybridization solution and then hybridized with the labeled probes. After several washes, the membranes were autoradiographed using a Gel imaging system (Amersham Imager 600, GE, Japan). U6 RNA was used a loading control. Probe sequences used for northern blot hybridizations are listed in Supplementary Table 1.

Northern blot analysis was conducted as described elsewhere (46 (link)). In short, the small RNA fraction was isolated from mock- or SFV-infected cells (MOI of 10, at 24 h p.i.) by using the mirVana miRNA isolation kit (Thermo Fisher Scientific). RNA was size fractioned on a 0.5× TBE–7 M urea–15% polyacrylamide gel, transferred to Hybond NX nylon membranes (GE Healthcare), and chemically cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma). Small RNAs were probed with a set of DNA oligonucleotides (Table S2) that were 5′ end-labeled with [γ-³²P]ATP (Perking-Elmer) using T4 polynucleotide kinase (New England Biolabs). Hybridization to the oligoprobes was performed overnight at 42°C in Ultrahyb Oligo hybridization buffer (Thermo Fisher Scientific). Membranes were washed twice at 42°C with each of the following three buffers: 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.5% SDS, 2× SSC and 0.2% SDS, and 0.2× SSC and 0.1% SDS. The membrane was exposed to a phosphorimager screen for signal detection.