Comparison of Three Magnetic Bead Surface Functionalities for RNA Extraction and Detection
Comparison of Three Magnetic Bead Surface Functionalities for RNA Extraction and Detection
复制标题
DOI：
10.1021/am506374t
复制
发表时间：
2015-03-25
影响因子：
9.5
通讯作者：
Haselton, Frederick R.
中科院分区：
文献类型：
--
作者：
Adams, Nicholas M.;Bordelon, Hali;Haselton, Frederick R.
磁珠可方便地在分子检测之前从生物样品中提取核酸生物标志物。这些珠子具有多种表面功能，旨在捕获特定的 RNA 子集。我们假设珠子表面功能会影响结合动力学、处理简单性以及与分子检测策略的兼容性。在本报告中，评估了三种旨在结合核酸、二氧化硅、标签 (dT) 和特定寡核苷酸序列的磁珠表面化学物质。市售的二氧化硅涂层珠子和寡聚 (dT) 珠子，以及用与呼吸道合胞病毒 (RSV) 核衣壳基因互补的寡核苷酸功能化的珠子，在掺有目标 RSV 特异性珠子的替代患者样品中孵育 1 分钟后，分别恢复类似于 71、类似于 71 和类似于 7% 目标 RSV mrNA，需要更长的孵育时间才能回收与其他珠子相当的目标量（类似于180 分钟时为 77%）。正如预期的那样，二氧化硅包被的珠子提取总 RNA，oligo (dT) 珠子选择性提取总 mRNA，RSV 特异性珠子选择性提取 RSV N 基因 mRNA。珠子功能的选择通常取决于目标检测策略。二氧化硅包被的珠子最适合需要 mRNA 以外的核酸的应用，特别是能够耐受高浓度非目标背景核酸的检测策略，例如 RT-PCR。另一方面，寡聚 (dT) 珠子最适合 mRNA 靶标，因为它们能快速结合生物标志物，具有相对较高的回收率，并且能够直接在珠子表面执行检测策略。序列特异性磁珠可能最适合不耐受高浓度非目标核酸的应用，这些非目标核酸需要不含 Poly(A) 尾的短 RNA 序列（例如 microRNA），或者直接在磁珠表面进行 RNA 检测。
Magnetic beads are convenient for extracting nucleic acid biomarkers from biological samples prior to molecular detection. These beads' are available with a Variety of surface functionalities, designed to capture particular Subsets of RNA. We hypothesized that bead surface functionality affects binding kinetics, processing simplicity, and Compatibility with molecular detection strategies. In this report, three Magnetic bead surface chemistries designed to bind nucleic acids, silica, tag (dT), and a specific oligonucleotide sequence were evaluated. Commercially available silica-coated and oligo (dT) beads, as Well as beads functionalized with oligonucleotides complementary to respiratory syncytial Virus (RSV) nucleocapsid gene, respectively recovered similar to 71, similar to 71, and similar to 7% target RSV mrNA after a 1 min of incubation time in a surrogate patient sample spiked with the target RSV specific beads required much longer incubation times to recover amounts of the target comparable to the other beads (similar to 77% at 180 min). As expected, silica-coated beads extracted total RNA, oligo (dT) bead selectively extracted total mRNA, and RSV-specific beads selectively extracted RSV N gene mRNA. The choice of bead functionality is generally dependent on the target detection strategy. The silica-coated beads are most suitable for applications that require nucleic acids other than mRNA, especially with detection strategies that are tolerant of a high concentration of nontarget background-nucleic acids, such as RT-PCR. On the other hand, oligo (dT) beads are best-suited for mRNA targets, as they bind biomarkers rapidly, have relatively high recovery, and enable detection strategies to be performed directly on the bead surface. Sequence-specific beads may be best for applications that are not tolerant of a high concentration of nontarget nucleic acids that require short RNA sequences without poly(A) tails, such as microRNAs, or that perform RNA detection directly on the bead surface.