Abstract
Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years, it has been aptly adapted to validate and study the size and expression of small noncoding RNAs. Here, we describe protocols employed in our laboratory for conventional northern analysis with total RNA/mRNA to study gene expression and validation of small noncoding RNAs using low molecular weight fraction of RNAs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019
Hsieh LC et al (2009) Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151:2120–2132
Zhu QH et al (2008) A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 18:1456–1465
Bo W et al (2009) Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv. Funct Integr Genomics 9:499–511
Lee H et al (2010) Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis. Nucleic Acids Res 38:3081–3093
Moldovan D et al (2009) Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis. J Exp Bot 61:165–177
Marin E et al (2010) miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. Plant Cell 22:1104–1117
Gao P et al (2010) Over-expression of osa-MIR396c decreases salt and alkali stress tolerance. Planta 231:991–1001
Priyanka B et al (2010) Characterization of expressed sequence tags (ESTs) of pigeon pea (Cajanus cajan L.) and functional validation of selected genes for abiotic stress tolerance in Arabidopsis thaliana. Mol Genet Genomics 283:273–287
Wu T et al (2010) Transcriptome profile analysis of floral sex determination in cucumber. J Plant Physiol 167:905–913
Zang Q et al (2010) Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat. J Biosci 35:379–388
Katiyar-Agrawal S et al (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci USA 103:18002–18007
Katiyar-Agarwal S, Gao S, Vivian-Smith A (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21:3123–3134
Katiyar-Agarwal S, Jin H (2007) Discovery of pathogen-regulated small RNAs in plants. In: Methods in enzymology, 427:215–227. Elsevier, Amsterdam
Pall GS et al (2007) 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
Beckmann BM et al (2010) Northern blot detection of endogenous small RNAs (∼14 nt) in bacterial total RNA extracts. Nucleic Acids Res 38:e147
Buhtz A et al (2010) Phloem small RNAs, nutrient stress responses, and systemic mobility. BMC Plant Biol 10:64
Xin M et al (2010) Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol 10:123
Lu et al (2008) Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs). Proc Natl Acad Sci USA 105:4951–4956
Kim SW et al (2010) A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Res 38:e98
Petersen M, Wengel J (2003) LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 21:74–81
Zou X et al (2010) Identification of transcriptome induced in roots of maize seedlings at the late stage of waterlogging. BMC Plant Biol 10:189
Jin H (2010) Screening of genes induced by salt stress from Alfalfa. Mol Biol Rep 37:745–753
Valdes-Lopez O (2010) MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity. New Phytol 187:805–818
Tang X et al (2007) A simple array platform for microRNA analysis and its application in mouse tissues. RNA 13:1803–1822
Meng L, Lemaux PG (2003) A simple and rapid method for nuclear run-on transcription assays in plants. Plant Mol Biol Rep 21:65–71
Schmittgen TD et al (2004) A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 32:e43
Shi R, Chiang VL (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39:519–525
Fu HG et al (2006) A novel method to monitor the expression of microRNAs. Mol Biotechnol 32:197–204
Chen C et al (2005) Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Res 33:e179
Varkonyi-Gasic E et al (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Jain M (2009) Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice. Plant Sci 176:702–706
Garg R et al (2010) Validation of internal Âcontrol genes for quantitative gene expression studies in chickpea (Cicer arietinum L.). Biochem Biophys Res Commun 396: 283–288
Herrin DL, Schmidt GW (1998) Rapid, reversible staining of northern blots prior to hybridization. Biotechniques 6:196–198
Acknowledgments
Research work in the laboratories of S.K.-A. and M.A. is supported by grants from Department of Biotechnology (DBT), India and University of Delhi. R.P. and A.R.B. are thankful to Council of Scientific and Industrial Research (CSIR), India and DBT, India for the student fellowship, respectively.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bhardwaj, A.R., Pandey, R., Agarwal, M., Katiyar-Agarwal, S. (2012). Northern Blot Analysis for Expression Profiling of mRNAs and Small RNAs. In: Jin, H., Gassmann, W. (eds) RNA Abundance Analysis. Methods in Molecular Biology, vol 883. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-839-9_2
Download citation
DOI: https://doi.org/10.1007/978-1-61779-839-9_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-838-2
Online ISBN: 978-1-61779-839-9
eBook Packages: Springer Protocols