Purification of pre-miR-29 by arginine-affinity chromatography

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• 作者：Patrí ; cia Pereira^a ; Â ; ngela Sousa^a ; Joã ; o Queiroz^a ; Ilí ; dio Correia^a ; Ana Figueiras^a ; ^b ; Fani Sousa^a ; ^{fani.sousa@fcsaude.ubi.pt" class="auth_mail}
• 关键词：miRNA ; microRNA ; RNA ; ribonucleic acid ; mRNA ; messenger RNA ; RISC ; RNA-induced silencing complex ; AD ; Alzheimer's disease ; A尾 ; 尾-amyloid ; APP ; 尾-amyloid precursor protein ; BACE1 ; 尾-site APP-cleaving enzyme 1 ; SPE ; silica-based solid-phase extraction ; PCR ; polymerase chain reaction ; cDNA ; complementary DNA ; AC ; affinity chromatography ; RNases ; RNA degrading enzymes ; tRNA ; transfer RNA ; sRNA ; small RNA.
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：1 March, 2014
• 年：2014
• 卷：951-952
• 期：Complete
• 页码：16-23
• 全文大小：761 K

文摘

Recently, differential expression of microRNAs, in patients with Alzheimer's disease (AD) suggests that they might have key regulatory roles in this neurodegenerative disease. Taking into account this fact, several studies demonstrated that the miR-29 is significantly decreased in AD patients, also displaying abnormally high levels of 尾-site APP-cleaving enzyme 1. Thus, RNA biochemical or structural studies often require a RNA sample that is chemically pure and biologically active. The present work describes a new affinity chromatography method using an arginine support to specifically purify pre-miR-29 from other Rhodovulum sulfidophilum small RNA species. Nevertheless, in order to achieve higher efficiency and selectivity, it is essential to characterize the behavior of pre-miR-29 binding/elution. Thus, three different strategies based on increased sodium chloride (280-500 mM), arginine (25 mM) or decreased ammonium sulfate (2-0.1 M) stepwise gradients are described to purify pre-miR-29. In this way, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (96.5 and 56.7%, respectively) was obtained. Moreover, the quality control analysis revealed high integrity in pre-miR-29 preparations as well as high purity (90 and 98%, respectively), demonstrated by the scarce detection of proteins. This improved method takes advantage of its simplicity, significant cost reduction, due to the elimination of some complex operations, and speed for large-scale purification of pre-miRNAs suitable for biochemical and structural studies.