Rediprime 2 random prime labelling system

Total RNA was extracted from Agrobacterium-infiltrated N. benthamiana or viroid inoculated S. tuberosum leaf tissues using the hot-phenol method as previously described13 (link). For high molecular weight RNA gel blots, 15 μg of total RNA was separated on 1.2% agarose gels containing 6% formaldehyde and transferred to nylon N⁺ membrane. For mature PSTVd and siPSTVd detection, the [a-³²P]-UTP-labelled T7 RNA transcripts from cDNA clones in pGEM-Teasy vector using the MAXIscriptkit (Ambion) were used as probes; for GFP mRNA detection, the full length GFP were radioactively labelled by [a-³²P]-dCTP using a Rediprime™II Random Prime Labelling System(GE healthcare), for siGFP detection, the [a-³²P]-UTP-labelled T7 RNA transcripts from pGEM-Teasy-GFP vector using the MAXIscriptkit (Ambion) were used as probes; for Virp1 mRNA detection, the full length NbVirp1 were radioactively labelled by [a-³²P]-dCTP using a Rediprime™II Random Prime Labelling System(GE healthcare); for miR167, miR159 and U6 detection, [r-³²P]ATP-labeled specific oligonucleotide probe sequences were used.
Dot-blot hybridization detection of PSTVd accumulation was performed as described (Monger WA et al. 2015). The PSTVd DNA fragment was labeled using the DIG DNA PCR labeling kit (Mylab Corporation).

Genomic DNA was isolated from whole seedlings, leaves or meristem-enriched tissue using the Illustra DNA Extraction Kit Phytopure (GE Healthcare). Total DNA was separated on 0.8% agarose gel, depurinated for 10 min in 250 mM HCl, denatured in 0.5 M NaOH and 1.5 M NaCl for 30 min, and neutralized in 0.5 M Tris, 1.5 M NaCl and 1 mM EDTA at pH 7.2 for 2×15 min. DNA was blotted onto Hybond N+ membranes (Amersham) with 20× SSC, washed and UV-crosslinked with a Stratalinker (Stratagene).
Hybridization was performed as described [56] (link). An ONSEN-specific probe (see Supplementary Table 2) was radioactively labelled with 50 µCi of dCT-α-³²P (Amersham) using the Rediprime II Random Prime Labelling System (GE Healthcare) and purified via G50 Probequant (Amersham) columns. Signals were detected using Phosphorimager screens (Amersham) and scanned by a Molecular Imager FX (Biorad). Densitometric quantification was performed using Image Lab software on three biological replicates.