Dig luminescent detection starter kit for nucleic acids

Northern blot analysis was performed essentially as previously described19 (link). Total RNAs were extracted from leaf tissues using TRIzol reagent (Invitrogen) and treated with DNase (TAKARA). 3 μg of total RNAs were separated on a denaturing 2% formaldehyde agarose gel and transferred to Hybond-N+ membranes (Amersham Bioscience) using 20 × SSC. The RNAs were cross-linked to the membrane matrix by UV for 45 s. Northern blotting for assays of CWMV genomic RNAs were carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). DNA oligonucleotides complementary to the 3′-terminus of the CWMV genome or specifically complementary to CWMV RNA1 were labeled with digoxigenin (DIG) at their 3′ ends using the DIG Oligonucleotide Tailing Kit (Roche) and then purified using a G25 Sephadex column (GE). Membranes were prehybridized for 2 h and hybridized overnight at 42 °C using the DIG Luminescent Detection Starter Kit for Nucleic Acids (Roche). The hybridization signals were visualized by the Amersham Imager 600 (GE). All these procedures followed the manufacturers’ instructions.

Northern blot assays were performed as previously described (Yang et al., 2016 (link)). Total RNAs were extracted from leaf tissues using TRIzol reagent (Invitrogen) and treated with DNase (TaKaRa). Three micrograms of total RNAs were separated on a denaturing 2% formaldehyde agarose gel and transferred to Hybond-N⁺ membranes (Amersham Bioscience) using 20× SSC buffer. The RNAs were cross-linked to membrane matrix by UV for 50 s. Northern blotting for assays of CWMV genomic RNAs was carried out using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). The DNA oligo nucleotides complementary to the 3′ terminus of the CWMV genome were labeled with digoxigenin (DIG) at their 3′ ends using the DIG Oligo nucleotide Tailing Kit (Roche) and then purified using a G25 Sephadex column (GE). Membranes were pre-hybridized for 2 h and hybridized overnight at 42°C using the DIG Luminescent Detection Starter kit for nucleic acids (Roche). The hybridization signals were visualized by Amersham Imager 600 (GE). All these procedures were performed according to the manufacturers’ instructions.