Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions

Cold Spring Harbor Protocols
Donald C Rio

Abstract

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.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·J C AlwineG R Stark
Sep 1, 1980·Proceedings of the National Academy of Sciences of the United States of America·P S Thomas
Jul 6, 2014·Cold Spring Harbor Protocols·Donald C Rio
Mar 4, 2015·Cold Spring Harbor Protocols·Timothy W Nilsen

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Citations

May 4, 2017·Cold Spring Harbor Protocols·Jo Ann Wise, Olaf Nielsen
Mar 4, 2015·Cold Spring Harbor Protocols·Timothy W Nilsen
Apr 4, 2015·Cold Spring Harbor Protocols·Timothy W Nilsen
Dec 17, 2021·Genome Biology·Maria Luz Annacondia, German Martinez

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