Plant total RNA was prepared by an SDS-phenol/chloroform extraction and a reverse transcription (RT) reaction was conducted as previously described [26 ,30 (link)]. BrYVs and PEMV 2 were detected using the primers BrYA484F/BrYB88F/BrYC257F/BrY761R and PEM2797F/PEM3202R, respectively. After PCR amplification, the products were separated in 1.5% agarose gels and stained with ethidium bromide.
Northern blot was performed as described [36 (link)]. RNAs used for BrYV (3 μg) and PEMV 2 (5 μg) were separated in a 1.2% formaldehyde-agarose gel and then transferred onto a Hybond-N+ nylon membrane. Prehybridization was performed for 5 h at 65 °C. The [α-32P] dCTP-labeled DNA probe specific for BrYV or PEMV 2 was generated using the Prime-a-Gene labeling system (Promega, Madison, WI, USA). Hybridization was carried out at 65 °C for 16 h. After washing, the nylon membrane was exposed to a storage phosphor screen (GE healthcare).
Northern blot was performed as described [36 (link)]. RNAs used for BrYV (3 μg) and PEMV 2 (5 μg) were separated in a 1.2% formaldehyde-agarose gel and then transferred onto a Hybond-N+ nylon membrane. Prehybridization was performed for 5 h at 65 °C. The [α-32P] dCTP-labeled DNA probe specific for BrYV or PEMV 2 was generated using the Prime-a-Gene labeling system (Promega, Madison, WI, USA). Hybridization was carried out at 65 °C for 16 h. After washing, the nylon membrane was exposed to a storage phosphor screen (GE healthcare).
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