Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.
In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA.
Donald C
DC
Rio
D015152:Blotting, Northern / Q000379:methods*; D002196:Capillaries; D004586:Electrophoresis; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification*
D012313:RNA