Northern Blotting of miRNA and mRNA

Northern blotting was carried out to examine the expression levels of miRNA and mRNA (19 (link)). For the miRNA Northern blotting, miRNAs were extracted from shrimp hemocytes using the mirVana miRNA isolation kit (Ambion, USA) according to the manufacturer's instructions. On the other hand, for Northern blot analysis of mRNA, total RNAs were extracted with RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, China). After electrophoresis for 2 h, the RNAs were transferred onto a nylon membrane (Amersham Biosciences, UK), followed by UV cross-linking. The membrane was pre-hybridized in DIG (digoxigenin) Easy Hyb granule buffer (Roche, Switzerland) for 0.5 h at 42°C and then hybridized with DIG-labeled miR-1000 probe (5′-TACTGCTGTGACGGGACAATAT-3′), U6 probe (5′-GGGCCATGCTAA TCTTCTCTGTATCGTT-3′), wsv191 probe (5′-TTCTTGGCTGCAGTTGAAACCC AGCGAACCCT-3′), wsv407 probe (5′-CTCTCCACCCTTTCAATGATGGTAATGG AAGAAC-3′) or β-actin probe (5′-ATGTCACGAACGATTTCTCGCTCGGCGGTG-3′) at 42°C overnight. In shrimp, β-actin is used as an internal control (19 (link)). The detection was done with the DIG High Prime DNA labeling and detection starter kit II (Roche).