Northern+Blotting

Northern blotting is useful for analyzing specific RNA sizes and for the determination of gene expression. Common in cell biology and biochemistry, the Northern Blot technique incorporates the use of RNA purification and gel electrophoresis to create a visualization of the RNA in question. It is useful for determining the presence of a specific sequence in multiple instances and for the identification of current gene expression. To determine the presence of specific RNA, the extraction and purification of nucleotides must occur. Separation of RNA from DNA and other nucleotides needs to be completed before gel electrophoresis separates the RNA strands by size. Figure 1. The basic Northern Blot method. Image copyright of Molecularstation.com
 * Description**

After purification and subsequent gel electrophoresis, the gel is placed onto a sheet of nitrocellulose or nylon paper [1]. Labeled DNA probes for a specific complementary sequence are added to a solution that is then used to incubate the paper. Post- incubation, the hybridized RNA can be visualized and size determined due to the position of the known labeled markers. Visualization can occur with either the naked eye or by a radiograph, depending on the type of label that was used. Gene expression is inferred by the presence of tRNA.

Taking already sorted by size RNA sequences, the technique lets an investigator visualize the specific sequence(s) of interest and determine the number of base pairs based on molecular weight. Gene expression can also be established by the presence of the RNA on the page, however it is time and cost intensive [2]. Figure 2. Developed nitrocellulose paper. Taken from []
 * Purpose**

Based on the Southern blot method, the Northern blot was created using RNA rather than DNA as the substrate. Developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University, it was named with respect to the method off of which it was based [3]. Since then it was used for the determination of gene expression along with relative gene mass.
 * Origin and History**

As gene study continues, so does the usage of Northern blotting. The usage of the technique is primarily used to determine the presence of a gene and gene regulation. In the case of “Suppression analysis of //esa1// mutants in //saccharomyces cerevisia// links //NAB3// to transcriptional silencing and nucleolar functions,” the technique was used in the determination of “//NAB3//-dependent changes in the //ESA1// transcript” [4]. Since over expression of //NAB3// did not change the transcription rates of the gene, it was found that the RNA transcript migrated due to a size change. Another recent study which included Northern blot analysis used it to show that the transcription level of a gene could be up-regulated during an infection [5]. This knowledge was then used to determine that the infected plant could serve as a positive control and thus letting the expression of virus-infected //Nicotiana benthamiana,// a different plant, be studied. A third recent studied used the technique to determine some virus-specific small RNAs in infected “//Ae. Albopictus// cell lines infected with wtBUNV” [6]. Without the use of “conventional” Northern blotting, the presence of the small RNA would not be able to be visually determined and the confirmation of the virus would not have been as prolific. Continuation of Northern blotting, then, is key for further virus and RNA change studies.
 * Recent Research**


 * References**
 * 1) Alberts, B., Johnson, A., Lewis, J. Raff, M., Roberts, K., Walter, P. 2008. Molecular Biology of the Cell, 5th ed. Garland Science, Taylor & Francis Group, NY, pp 538-539.
 * 2) Schlamp, K., Weinmann, A., Krupp, M., Galle, P. R., & Teufel, A. (2008). BlotBase: A northern blot database.  //Gene//, 427(2): 47-50.
 * 3) Alwine, J. C., Kemp, D. J., & Stark, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. //Proceedings of the National Academy of Sciences of the USA//, 74(12): 5350-5354.
 * 4) Chang, C. S., Clarke, A., & Pillus, L. (2012). Suppression analysis of //ESA1// mutants in //saccharomyces cerevisia// links //NAB3// to transcriptional silencing and nucleolar functions. //G3//, 2(10): 1223-1232.
 * 5) Liu, D., Shi, L., Han, C., Yu, J., Li, D., & Zhang, Y. (2012) Validation of reference genes for gene expression studies in virus-infected //Nicotiana benthamiana// using quantitative real-time PCR. //PLoS One//, 7(9): e46451.
 * 6) Szemiel, A. M., Failloux, A., Elliot, R. M. (2012). Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells. //PLoS Neglected Tropical Diseases// 6(9): e1823.