A sensitive non-radioactive northern blot method to detect small RNAs.
The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.
Kim
Sang Woo
SW
Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
Li
Zhihua
Z
Moore
Patrick S
PS
Monaghan
A Paula
AP
Chang
Yuan
Y
Nichols
Mark
M
John
Bino
B
D015152:Blotting, Northern / Q000379:methods*; D002021:Buffers; D002460:Cell Line; D004076:Digoxigenin; D005022:Ethyldimethylaminopropyl Carbodiimide; D006801:Humans; D035683:MicroRNAs / Q000032:analysis; D015345:Oligonucleotide Probes / Q000737:chemistry; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D022661:RNA, Untranslated / Q000032:analysis*; D013696:Temperature
D002021:Buffers; D035683:MicroRNAs; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D022661:RNA, Untranslated; C477371:locked nucleic acid; D004076:Digoxigenin; D005022:Ethyldimethylaminopropyl Carbodiimide