Cdp star chemiluminescent substrate for alkaline phosphatase

Northern blot was performed as previously described with slight modifications [35 (link)]. In brief, 20 μg of total RNA extracted from infected cells was separated on a 15 % denaturing PAGE (7.5 M urea). The sample was transferred to positively charged nylon membranes (Roche, Mannheim, Germany) and cross-linked to the membranes through UV irradiation (120 mJ/cm²) [36 (link)]. After pre-hybridization in Ultrasensitive Hybridization Buffer (Ambion, Life technologies, USA) for more than 0.5 h at 37 °C, blots were hybridized overnight at 37 °C with 0.5 nM miRCURY LNA™ probes modified both by 5’ digoxigenin (DIG) and 3’ DIG (Exiqon, Vedbaek, Denmark). Afterward, the blots were sequentially washed using low-stringency, high-stringency, and washing buffers at 37 °C and then incubated in blocking buffer (Roche, Mannheim, Germany) for 3 h. The blots were subsequently incubated in blocking buffer with an anti-DIG-AP Fab fragment solution (Roche, Mannheim, Germany) for 0.5 h and then washed in DIG washing buffer (Roche, Mannheim, Germany). The blots were visualized through autoradiography using a CDP-Star chemiluminescent substrate for alkaline phosphatase (Roche, Mannheim, Germany).

Genomic DNA samples (10–21 µg) from candidate clones and unmanipulated controls were digested with SacI and HindIII (Fermentas) restriction endonucleases, separated on 0.8% agarose gels, blotted onto Hybond N+ membranes (Amersham), and hybridized with a PCR Dig-labeled pDM576 probe (primers: 5’-GCTAGGAAGCAGCCAATGAC-3’ and 5’-CATTCCCGGCTACAAGGAC-3’), as previously described^{14 (link)}. Detection of DNA fragments was carried out using CDP-Star Chemiluminescent Substrate for Alkaline Phosphatase (Roche).