Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot.
The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20-40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25-50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.
Pall
Gurman Singh
GS
Division of Cancer Sciences and Molecular Pathology, Glasgow University, Western Infirmary, Dumbarton Road, Glasgow, G11 6NT, UK.
Codony-Servat
Carles
C
Byrne
Jane
J
Ritchie
Leigh
L
Hamilton
Andrew
A
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D002234:Carbodiimides; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D009757:Nylons / Q000737:chemistry; D015345:Oligonucleotide Probes; D018749:RNA, Plant / Q000032:analysis; D034741:RNA, Small Interfering / Q000032:analysis*; D051381:Rats; D013696:Temperature; D014466:Ultraviolet Rays
C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D035683:MicroRNAs; D009757:Nylons; D015345:Oligonucleotide Probes; D018749:RNA, Plant; D034741:RNA, Small Interfering