Northern Blotting for RNA Detection

RNA was extracted with a NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. Northern blotting was performed as previously described (39 (link)). In brief, 0.2 μg of RNA was denatured and separated by an 0.8% agarose-formaldehyde gel at 70 V for 5 h. The agarose gel was soaked in 50 mM NaOH for 50 min to break the large RNA fragment. Subsequently, the gel was washed with 100 mM Tris-HCl (pH 7.5) for 30 min and incubated in 20× SSC buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 20 min and then capillary transferred to a positively charged Hybond-N nylon membrane (Amersham Biosciences) overnight. RNA was immobilized by UV cross-linking (1,800 × 100 μJ/cm) and hybridized at 50°C overnight with digoxigenin (DIG)-labeled probes generated with a PCR DIG probe synthesis kit (Roche Diagnostics). The primers used to synthesize the DIG-labeled nCoV19-N cDNA probe were 5′-AAGCTGGACTTCCCTATGGTGC-3′ and 5′-CCTTGGGTTTGTTCTGGACCACG-3′. The probes of porphobilinogen deaminase (PBGD) used as the internal control in Northern blotting were 5′-GGTGACCAGCACACTTTGGG-3′ and 5′-AGCCGGGTGTTGAGGTTTCC-3′.