Focus on RNA isolation: Obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue

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.

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

“…This regulation may be another example of ''downstream'' miR-124 related mechanisms. miR-128 is another miRNA strongly concentrated in the brain relative to other organs (Lagos-Quintana et al 2002;Baskerville and Bartel 2005) and enriched in cultured primary rat neurons relative to primary rat astrocytes (Smirnova et al 2005;Wang et al 2008b). Interestingly, there is an increase in miR-128 expression in the Alzheimer's disease hippocampus relative to control hippocampus (Lukiw 2007).…”

“…H4 cells were isolated from a brain tumor (Arnstein et al 1974) and express a miRNA profile that more closely resembles cultured astrocytes than cultured neurons (Wang et al 2008b). These cells have various genetic anomalies (Krex et al 2001).…”

“…Figure 4B shows the results of miRNA microarray study of H4 glioneuronal cells showing relative expression level of miR-107, miR-124, miR-128, and miR-320 under normal culture conditions. These arrays were performed in triplicate on biological replicates exactly as previously described (Wang et al 2008b). MiR-320 is highest expressed, miR-128 is lowest expressed, and the difference between miR-107 and miR-124 expression is not statistically significant in the cells.…”

Bead Array–Based microRNA Expression Profiling of Peripheral Blood and the Impact of Different RNA Isolation Approaches

“…26 -28 Technical issues regarding high-throughput miRNA expression profiling are discussed by various authors. 29,30 With regard to peripheral blood, most of these studies were based on RNA extracted from either whole blood or extracted peripheral blood mononuclear cells (PBMCs).…”

Development of the “Three-step MACS”: a Novel Strategy for Isolating Rare Cell Populations in the Absence of Known Cell Surface Markers from Complex Animal Tissue

“…Analyzing whole organs or tissues often introduces irrelevant cell types into the sample, which distorts the data (e.g., gene expression profiles) of all of the cell types, which, on average, are obtained, rather than that of any particular cell type. [5][6][7][8] Microdissected samples are purer, but they are laborious and technically challenging to prepare; hence, sample quantities are small. 9 Nucleic acid amplification is often needed downstream, a step that introduces bias.…”