Northern blot hybridization

Traditionally, Northern blot analysis of isolated total RNA or mRNA has been used to determine differences in levels of mRNA between and among different animal groups, tissues or time-points. Standard protocols for performing Northern blot hybridization are available (Sambrook and Russell, 2001). Radioisotopic and colorimetric methods are available for probe labeling. The advantage of Northern blot analysis, especially... 

Whereas mieroarray analysis of gene expression employs similar steps as Northern blot hybridization, it is radically different in principle. The major difference is that the roles of the nucleie aeid bound to the substrate and the labeled nueleic acid in the hybridization solution are reversed for mieroarrays. In mieroarray analysis, the DNA probe is fixed to the substrate with eaeh spot on the array con-... 

Northern Blot Hybridizations. Total or polyA+RNAs were denatured with dimethyl sulfoxide and glyoxal, size fractionated on 1% agarose gels, and transferred to nitrocellulose filters. Labeled DNA probes were denatured by boiling and hybridized to filters for 16 hr at the appropriate temperature in 50% formamide, 25 mM phosphate buffer pH 6.8, 5X SET (IX SET - 150 mM NaCl, 20 mM Tris pH 7.8, 1 mM EDTA), 0.1% sodium dodecylsulfate (SDS), 10% dextran sulfate,... 

Isolation of RNA The R rubrum cells were broken by repeated freezing in liquid and thawing and the RNA was obtained in the aqueous phase after successive extraction with phenol-chloroform and chloroform-1soamyl alcohol(8). Northern blot hybridization and RNA dot blot hybridization Both were performed as in (7,9). 

Because a single size band may contain several DNA species, several clones should be sequenced so that the real differentially displayed clones are not missed. If several sequences appear, individual clones should be tested as probes in Northern blot analysis, as described below. An alternative strategy has been described to clone specific products after Northern blot hybridization. This procedure involves the capturing and the reampfification of the hybridized cDNA from the membrane (13). 

Fig. 2. Correlation between pCNT103 mRNA accumulation (—) and cell division (...) after treatment of hormone-starved cells with various 2.4-D concentrations. Cell density was determined at the start of the experiment (t = 0) and 72 h after hormone addition. The increase in cell density is given as Incells/ml (t = 72) — In cells/ml (t = 0). The level of pCNT103 mRNA was determined 4 h after hormone treatment by Northern blot hybridization and densitometric scanning of the autoradiographs [Adapted from 17]...

This antisense strategy has been used to investigate the role of CRABP I and CRABP II in RA-induced gene expression. Alterations m the expression of RAR P, TGF-p3, and tenascin are determined by Northern-blot hybridization using total-cellular RNA. 

Steady-state levels of mRNA are determined by Northern-blot hybridization. 

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