Objective: With the application of high-throughput sequencing methods, more and more sRNAs are required to be verified. In this study, we developed the digoxigenin-labeled Northern blot method for detection of Yersinia pestis sRNA.
Methods: Total RNAs extracted from Yersinia pestis grown under low-iron conditions were loaded onto 10% denaturing urea polyacrylamide gel (dPAGE), electrophoresed and transferred to nylon membranes. Northern blots were fixed to the membrane by UV cross-linking and subjected to hybridization with 3' -end digoxigenin-labeled oligonucleotides RNA probe for RyhB1 and RyhB2 overnight and followed by washing, blocking, immunological detection and finally exposed to film.
Results: Exposure time of digoxigenin-labelled Northern blot was 20s-3 min. The detection sensitivity of RyhB1 and RyhB2 was 0.005 microg and 0.05 microg, respectively. RyhB1 and RyhB2 probe specificity was high and no cross reaction with each other was found. Positively charged or neutral nylon membranes are applicable to the hybridization reaction. Hybridization for RNA probe can proceed within 42 to 65 degrees C with reduced non-specific probe interactions by increasing the temperature while the hybridization temperature for DNA oligonucleotide probes seemed to be determined empirically.
Conclusion: A digoxigenin-labeled Northern blot has been developed for detection of Yersinia pestis sRNA, which was characterized by good specificity, high sensitivity, longer shelf life and short exposure times and provided an power tool for validation of bacterial sRNAs and function studies.