Improved northern blot method for enhanced detection of small RNA

Nature Protocols

Volumn 3, Issue 6, 2008, Pages 1077-1084

  Pall, Gurman S ^a   Hamilton, Andrew J ^a  

^a UNIVERSITY OF GLASGOW   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

1 (3 DIMETHYLAMINOPROPYL) 3 ETHYLCARBODIIMIDE; MICRORNA; PIWI PROTEIN; SMALL INTERFERING RNA;

ANIMAL CELL; ARTICLE; CONTROLLED STUDY; CROSS LINKING; DENATURING GRADIENT GEL ELECTROPHORESIS; EMBRYONIC STEM CELL; GENE TRANSFER; GENETIC MARKER; IMAGE ENHANCEMENT; IMAGING; MOUSE; NONHUMAN; NORTHERN BLOTTING; NUCLEIC ACID ANALYSIS; NUCLEIC ACID PROBE; PRIORITY JOURNAL; PROTEIN FOLDING; RNA EXTRACTION; RNA HYBRIDIZATION; RNA ISOLATION; SENSITIVITY ANALYSIS; SIGNAL DETECTION;

ANIMALS; BLOTTING, NORTHERN; CROSS-LINKING REAGENTS; EMBRYONIC STEM CELLS; MICE; MICRORNAS; PHOSPHORUS RADIOISOTOPES; RNA, SMALL INTERFERING;

EID: 44949098384     PISSN: 17542189     EISSN: 17502799     Source Type: Journal    
DOI: 10.1038/nprot.2008.67     Document Type: Article

References (19)

1. Jiang, J., Lee, E.J., Gusev, Y. & Schmittgen, T.D. Real-time expression profiling of microRNA precursors in human cancer cell lines. Nucleic Acids Res. 33, 5394-5403 (2005).
2. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415-419 (2003).
3. Berezikov, E., Cuppen, E. & Plasterk, R.H. Approaches to microRNA discovery. Nat. Genet. 38 (suppl.): S2-S7 (2006).
4. Lee, Y., Jeon, K., Lee, J.T., Kim, S. & Kim, V.N. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21 4663-4670 (2002).
5. Church, G.M. & Gilbert, W. Genomic sequencing. Proc Natl. Acad. Sci. USA 81, 1991-1995 (1984).
6. Saito, I., Sugiyama, H., Furukawa, N. & Matsuura, T. Photoreaction of thymidine with primary amines. Application to specific modification of DNA. Nucleic Acids Symp. Ser. 61-64 (1981).
7. Khandjian, E.W. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol. Biol. Rep. 11, 107-115 (1986).
8. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200 (2001).
9. Vagin, V.V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320-324 (2006).
10. Yang, Z., Ebright, Y.W., Yu, B. & Chen, X. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′-terminal nucleotide. Nucleic Acids Res. 34, 667-675 (2006).
11. Ghosh, S.S. & Musso, G.F. Covalent attachment of oligonucleotides to solid' supports. Nucleic Acids Res. 15, 5353-5372 (1987).
12. Gingeras, T.R., Kwoh, D.Y. & Davis, G.R. Hybridization properties of immobilized nucleic acids. Nucliec Acids Res. 15, 5373-5390 (1987).
13. Godfroid, E., Min Hu, C., Humair, P.F., Bollen, A. & Gem, L. PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease.J. Clin. Microbiol. 41, 3690-3698 (2003).
14. Pall, G.S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007).
15. Sambrook, J. & Russell, D.W. Molecular Cloning. A Laboratory Manual 3rd edn. Vols. 1-3 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
16. Chomczynski, P. & Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581-585 (2006).
17. Hamilton, A.J. & Baulcombe, D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952 (1999).
18. Gy, I. et al. Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 19, 3451-3461 (2007).
19. Chomczynski, P. Solubilization in formamide protects RNA from degradation. Nucleic Acids Res. 20, 3791-3792 (1992).