Abstract
Investigation of gene expression significantly contributes to our knowledge of the regulation and function of genes in many areas of biology. In this protocol, we describe how northern blot analysis is used to identify gene expression patterns at the RNA level in human cancer cells as well as in cancerous and normal tissues. RNA molecules are separated by gel electrophoresis and are subsequently transferred to a porous membrane by capillary action. Specific sequences in the RNA are detected on the membrane by molecular hybridization with radiolabeled nucleic acid probes. Despite the development of newer methods, such as real-time PCR, nuclease protection assays and microarrays, northern blot analysis is still a standard technique used in the detection and quantification of mRNA levels because it allows a direct comparison of the mRNA abundance between samples on a single membrane. This entire northern blotting protocol takes ∼4 d to complete.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alwine, J.C., Kemp, D.J. & Stark, G.R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354 (1977).
Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159 (1987).
Welsch, T., Kleeff, J. & Friess, H. Molecular pathogenesis of pancreatic cancer: advances and challenges. Curr. Mol. Med. 7, 504–521 (2007).
Kleeff, J. et al. Pancreatic cancer: from bench to 5-year survival. Pancreas 33, 111–118 (2006).
Ramsay, G. et al. DNA chips: state-of-the art. Nat. Biotechnol. 16, 40–44 (1998).
O'Driscoll, L. et al. The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells. Cytotechnology 12, 289–314 (1993).
Friess, H. et al. Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer. Cell Mol. Life Sci. 60, 1180–1199 (2003).
Friess, H. et al. Identification of disease-specific genes in chronic pancreatitis using DNA array technology. Ann. Surg. 234, 769–778 (2001); discussion 778–779.
Grutzmann, R. et al. Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes. Oncogene 24, 5079–5088 (2005).
Friess, H. et al. Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression. Cancer Res. 53, 2704–2707 (1993).
Friess, H. et al. Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology 105, 1846–1856 (1993).
Korc, M. et al. Overexpression of the epidermal growth factor receptor in human pancreatic cancer is associated with concomitant increases in the levels of epidermal growth factor and transforming growth factor alpha. J. Clin. Invest. 90, 1352–1360 (1992).
Ramadori, G., Sipe, J.D. & Colten, H.R. Expression and regulation of the murine serum amyloid A (SAA) gene in extrahepatic sites. J. Immunol. 135, 3645–3647 (1985).
Chirgwin, J.M. et al. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299 (1979).
Hodgson, C.P. & Fisk, R.Z. Hybridization probe size control: optimized 'oligolabelling'. Nucleic Acids Res. 15, 6295 (1987).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13 (1983).
Feinberg, A.P. & Vogelstein, B. 'A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity'. Addendum. Anal. Biochem. 137, 266–267 (1984).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Streit, S., Michalski, C., Erkan, M. et al. Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues. Nat Protoc 4, 37–43 (2009). https://doi.org/10.1038/nprot.2008.216
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.216
- Springer Nature Limited
This article is cited by
-
Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes
BMC Genomics (2022)
-
PTT-quant: a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches
Applied Microbiology and Biotechnology (2022)
-
Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters
Microchimica Acta (2019)
-
Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation
Scientific Reports (2017)
-
Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation
Nature Communications (2016)