Nytran nx membrane

For Northern blot analyses, 4–5 µg of total RNA was separated on formaldehyde-1.2% agarose gels and blotted to a Nytran NX membrane (GE Healthcare, Chicago, IL, USA) by the capillary method using 20xSSC. Hybridization was carried out at 65 °C in Church buffer (0.5 M phosphate buffer, pH 7.2 containing 1% BSA, 1 mM EDTA, 7% SDS) overnight with the appropriate radioactively labelled probe, washed for 5 min in 2 × SSC, 0.1% SDS and for 15 min in 0.5 × SSC, 0.1% SDS at the hybridization temperature and exposed to an X-ray film. AHVd specific, P32-labelled DNA probes were prepared by using the Thermo Scientific DecaLabel DNA labelling kit (Thermo Fisher). As a template, we used the PCR-amplified and purified product of a cloned viroid.

For Northern blot analysis, 2–4 µg of total RNA was separated on formaldehyde/1.2% agarose gels and blotted to Nytran NX membrane (GE Healthcare, Chicago, IL, USA) via the capillary method using 20× SSC. Hybridization was performed at 65 °C in Church buffer (0.5 M phosphate buffer, pH 7.2, containing 1% BSA, 1 mM EDTA, and 7% SDS) overnight with the appropriate radioactively labeled probe, washed for 5 min in 2× SSC and 0.1% SDS, and incubated for 15 min in 0.5× SSC and 0.1% SDS at the hybridization temperature and exposed to X-ray film. Virus-specific P32-labeled DNA probes were prepared using the Thermo Scientific Decalabel DNA labeling kit.

For the small RNA Northern blot analyses, the total RNA samples (5 µg) were fractionated on denaturing 12% polyacrylamide gels, which contained 8 M urea, and were transferred to Nytran NX membrane (GE Healthcare, Chicago, IL, USA) via a capillary method using 20xSSC, and were fixed by ultraviolet cross-linking. Membranes were probed with radioactively 32P-labeled LNA oligonucleotide probes (Exiqon, Vedbæk, Hovedstaden, Denmark), which were complementary to the mature microRNAs (Table S6), using a published protocol [38 (link)]. Briefly, 5 pmol of each oligonucleotide probe was end-labeled with [γ32P] ATP by using T4 polynucleotide kinase. The prehybridization of the filters was carried out in 50% formamide, 0.5% SDS, 5xSSPE, 5x Denhardt’s solution, and 20 µg/mL of sheared denatured salmon sperm DNA. The hybridization was carried out at 50 °C for 2 h in the same buffer. The washing of the membranes was performed for 10 min, two times, with a washing solution containing 0.1%SDS and 2xSSC at the hybridization temperature.