Northern analysis

RDM provides the best results with mRNAs that are highly expressed, preferably from their own promoter. We observed a decrease in the quality of the results when using reporter mRNAs (e.g., GFP and luciferase) that are not native to the organism (yeast, in our case). The decrease in quality was apparent in the northern analyses, in which multiple bands or smears were observed. This is probably due to inefficient processing of these exogenous transcripts. 

The two human-cell forms of FcyRIII are encoded for by two separate genes, FcyRIIIA and FcyRIIIB a single mRNA size of 2.2 kb is detected in Northern analyses. The genes are mapped to chromosome 1, and FcyRII and FcyRIII are located within 200 kb of the genome. The existence of two... 

In both Southern and Northern analyses restriction-digested DNA fragments, mRNA, and polyA mRNA are separated by size when electrophoresed on agarose gel. The separated molecules are transferred, by electroblotting or capillary blotting, on to a nylon or nitrocellulose membrane. The immobilized RNA or DNA is reacted with a radiolabeled, chemiluminescent, or fluorescent probe that is complementary to the DNA/RNA of interest, unbound probe is washed off, and the membrane exposed, in the case of radioactive probes, to radioautographic film to visualize the sample of interest. 

The conversion of choline to choline-P was first demonstrated in yeast extracts by Wittenberg and Komberg (more famous for his contributions to DNA replication) in 1953. The enzyme was purified (K. Ishidate, 1984) from rat kidney and shown also to phosphorylate ethanolamine [5]. This kinase is now referred to as choline kinase p. Three isoforms of choline kinase have been identified (al oi2, and P). Northern analyses indicate that the mRNA encoding choline kinase al is most abundant in testis. Choline kinase o2 is a splice variant of choline kinase al. The choline kinase a and P genes (Chka and Chkb, respectively) have been characterized. The length of Chka is 40 kb whereas Chkb is only... 

The following figures show results of Northern analyses of poly(A) RNA prepared from various rat tissues and hybridized with different probes. In Fig. 7, the structure coding sequence of vl is illustrated by the bar at the top with the filled portion designating sequences in common with the receptor to the breakpoint shown by a triangle therefore, probe A used in... 

Northern analyses of RNA isolated from various organs of maize identified the gl8 mRNA as a 1.4 kb band (Fig. 3). In homozygous gl8/gl8 seedlings the accumulation of the gl8 mRNA is reduced to between 1/3 and 1/5 of the level in found in wild-type seedlings (Fig. 3). In wild-type plants the gl8 mRNA is detectable in all organs examined, however, maximum accumulation occurs in seedling tissue (Fig. 3). 

Fig. 2. Northern analysis of pgaX expression using a 2.0 kb pgaX Pst fragment as a probe. A. tubingensis was pregrown for 20 h on 1% sucrose and transferred to medium with different carbon sources. Lane 1 1% glucose lane 2 2% glucose lane 3 1% galacturonic acid lane 4 1% galacturonic acid, 1% glucose.

Figure 5A,B. Northern analysis of an A. aculeatus multicopy transformant (A) compared to the wild type (B). RNA was isolated from the mycelium before (lanes 1), 6 h after (lanes 2) and 24 h after (lanes 3) transfer of the corresponding strains to minimal medium (MM) with 1 % apple pectin. The location of the rhgA transcript is indicated with an arrow.

Multiple ODNs complementary either to the same transcript or to different transcripts can be used in the same reaction. If several ODNs to the same transcript are used, partial products are also expected (i.e., cleavage by only one ODN Fig. 9.2). Thus, the cleavage plan should be such that the full cleavage products and the various partial cleavage products are of distinct sizes. In cases where there is no good separation in size, it is possible to design probes that will recognize only some of the products in the northern analysis. 

Dry the pellet and resuspend in 10 pi of sterile, RNAse-free water. Keep at —20°. Take about half of the sample to northern analysis. 

The sedimentation of RDM cleavage products is determined by standard northern analysis (Fig. 9.3, step 7). Any northern protocol can be used for this purpose. The protocol presented here is widely used... 

The methods for genome-wide analysis of ribosomal association are conceptually different from those for analysis of a single mRNA (e.g., northern analysis). While in assays of a single gene by northern analysis it is simple to compare the distribution of an mRNA in different fractions, in a genome-wide assay, the mRNAs are usually compared relative to others within the fraction. Because of that, it is important to analyze by northern analysis several mRNAs that appeared to be affected to different extents, and to compare the relative effects among this group between the microarrays and the northern analysis. 

In experiments where spike-in controls are added, validation is simpler because one can perform a northern analysis for one of the spiked-in RNAs and use their signals to normalize any differences between fractions. This allows direct comparison between northern blot and microarray results. 

Northern analysis of transformants under anaerobic conditions... 

Ectopic expression of growth factors and autostimulation are often responsible for factor independence. As shown above, CSF-1 secreted by MSS cells may be a potent stimulator of at least short-term proliferation of Myl-D7 cells. We therefore compared the steady-state CSF-1 mRNA levels of uncloned Myl-D7 cells, of several stroma-dependent clones and of many stroma-independent clones by Northern analysis (Fig. 9), to analyze if CSF-1 is expressed in stroma-independent Myl-D7 mutants. 

Northern analysis is usually used to identify and quantitate specific mRNAs in a sample. Southern analysis is used to determine whether or not a gene of interest is present as well as its copy number. Other uses for Southern analysis include identifying restriction fragment length polymorphisms and changes in heterozygosity. 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

The first identified cannabinoid receptor subtype, CB was cloned and demonstrated to have an amino acid sequence consistent with a tertiary structure typical of the seven transmembrane-spanning proteins that are coupled to G proteins. In addition to being found in the central nervous system, mRNA for CB has also been identified in testes. The central nervous system responses to cannabinoid compounds are believed to be mediated exclusively by CB, inasmuch as CB2 transcripts could not be found in brain tissue by either Northern analysis or in situ hybridization studies. CBj transduces signals in response to central-nervous-system-active constituents of C. sativa as well as synthetic bicyclic and tricyclic cannabinoid analogs, aminoalkylindole, and eicosanoid cannabimimetic compounds. CB is coupled to G, to inhibit adenylate cyclase activity and to a pertussis-sensitive G protein to regulate Ca2+ currents. 

Fig. 2B. Northern analysis of poly A( + ) mRNA from chick duodenum hybridized to a calbindin-D2BK cDNA probe showing 3 mRNA species at 2100, 2800 and 3100 nucleotides.

Northern analysis of RNA from cyc S49 cells (Fig. 11) shows absence of as coding sequences, in agreement with the absence of as polypeptides in these cells. The levels of as mRNA in different tissues is variable. Very likely, as subunits are under developmental and hormonal control and experiments exploring this should be forthcoming shortly. 

Fig. 11. Northern analysis of RNA fractions isolated from tissues and cell lines for presence of mRNA coding for a-subunits of Gs. Note absence of signal in S49 cyc cells and that levels of a, mRNAs are not constant. Immature and mature rats were 10 and 180 days, respectively. (From Mattera et al. [ 15]).

Figure 2.7. Generalized procedure for Southern and Northern analysis. [Adapted from Vol-gelstein, B., and Kinzler, K. W. (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998.]...

H6. Harper, M. E., Goddard, L., Glynne-Jones, E., Peeling, W. B., and Griffiths, K., Epidermal growth factor receptor expression by northern analysis and immunohistochemistry in benign and malignant prostatic tumors. Eur. J. Cancer. 31A, 1492-1497 (1995). 

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