Focus on RNA Isolation: Obtaining RNA for MicroRNA (miRNA) Expression Profiling Analyses of Neural Tissue

Authors

Wang-Xia Wang, University of KentuckyFollow
Bernard R. Wilfred, University of KentuckyFollow
Donald A. Baldwin, University of Pennsylvania
R. Benjamin Isett, University of Pennsylvania
Na Ren, University of Kentucky
Arnold J. Stromberg, University of KentuckyFollow
Peter T. Nelson, University of KentuckyFollow

Abstract

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.

Document Type

Article

Publication Date

11-2008

Notes/Citation Information

Published in Biochimica et Biophysica Acta -- Gene Regulatory Mechanisms, v. 1779, issue 11, p. 749-757.

Copyright © 2008 Elsevier B.V.

© 2008. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/.

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.bbagrm.2008.01.005

Funding Information

Funding was provided through NIH K08 NS050110.

Repository Citation

Wang, Wang-Xia; Wilfred, Bernard R.; Baldwin, Donald A.; Isett, R. Benjamin; Ren, Na; Stromberg, Arnold J.; and Nelson, Peter T., "Focus on RNA Isolation: Obtaining RNA for MicroRNA (miRNA) Expression Profiling Analyses of Neural Tissue" (2008). Sanders-Brown Center on Aging Faculty Publications. 101.
https://uknowledge.uky.edu/sbcoa_facpub/101