title: Simple and nonradioactive detection of microRNAs using digoxigenin (DIG)-labeled probes with high sensitivity.
abstract: The discovery of microRNAs (miRNAs), which are ∼21-23 nucleotides that can regulate targeted mRNA by transcript cleavage or protein translation suppression, has changed the landscape of biomedical field greatly. At present, Northern blot analysis based on radioisotopes is still the most popular method on the detection of miRNAs for its high sensitivity. However, radioisotopes have been known for certain disadvantages, such as instability, expense, and safety; thus, developing a nonradioactive and highly sensitive method is needed. Here, we report a simple, nonradioactive, and sensitive method for miRNAs detection based on 5'-phos-3'-DIG-labeled probes prepared through splinted ligation and EDC cross-linking (DSLE). The method was more sensitive than traditional Northern blots with a DIG-labeled DNA probe and can detect as low as 2 fmol of miRNAs. The whole procedure can be completed within 6-8 h. DSLE method is very convenient, cost-effective, time-saving, and highly sensitive.
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Pengtao
authors.initials: P
authors.lastname: Gong
authors.forename: Jianhua
authors.initials: J
authors.lastname: Li
authors.forename: Ju
authors.initials: J
authors.lastname: Yang
authors.forename: Guocai
authors.initials: G
authors.lastname: Zhang
authors.forename: He
authors.initials: H
authors.lastname: Li
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Yang
authors.forename: Xichen
authors.initials: X
authors.lastname: Zhang
mesh_terms: D015152:Blotting, Northern; D015342:DNA Probes; D004076:Digoxigenin / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; D004076:Digoxigenin
keywords: DIG; EDC cross-linking; Northern blots; miRNAs; splinted ligation