AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.
AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.
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AFBI 测定 - 适用于培养的贴壁细胞的适体荧光结合和内化测定。
DOI：
10.1016/j.ymeth.2016.03.005
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Giangrande,PalomaH
中科院分区：
文献类型：
--
作者：
Thiel,WilliamH;Giangrande,PalomaH
SELEX（指数富集配体系统进化）过程可以从复杂的核酸库中富集特定于目标分子的 DNA 或 RNA 适体。 SELEX 过程已从使用重组靶蛋白在体外鉴定适体到基于细胞的方法 (Cell-SELEX) 进行了调整，其中靶标在细胞表面表达。 Cell-SELEX 的一大优势是目标分子保持天然确认。此外，Cell-SELEX 可用于通过对患病细胞与健康细胞进行选择来发现新型治疗生物标志物。然而，Cell-SELEX 需要注意的是，对选择中确定的单个适体进行测试是费力、耗时且昂贵的。筛选适体在细胞上的结合和内化的最常用方法是流式细胞术和定量 PCR (qPCR)。虽然流式细胞术可以直接评估荧光标记适体与靶标的结合，但它需要大量的起始材料并且不易扩展。基于 qPCR 的方法灵敏度很高，但由于样品处理步骤的数量，实验间的变异性不可忽略。在此，我们描述了基于细胞的适体荧光结合和内化（AFBI）测定。该测定需要最少的试剂，并且实验步骤/操作很少，从而可以同时快速筛选许多适体和条件，并直接定量适体结合和内化。
The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamersin vitrousing recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization.
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影响因子：
14.9
作者：
Thiel KW;Hernandez LI;Dassie JP;Thiel WH;Liu X;Stockdale KR;Rothman AM;Hernandez FJ;McNamara JO 2nd;Giangrande PH
通讯作者：
Giangrande PH
DOI：
10.1007/978-1-4939-1538-5_11
发表时间：
2015
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Thiel, William H;Thiel, Kristina W;Flenker, Katie S;Bair, Tom;Dupuy, Adam J;McNamara, James O 2nd;Miller, Francis J;Giangrande, Paloma H
通讯作者：
Giangrande, Paloma H
DOI：
10.1073/pnas.95.6.2902
发表时间：
1998-03-17
影响因子：
11.1
作者：
Morris, KN;Jensen, KB;Gold, L
通讯作者：
Gold, L
影响因子：
56.9
作者：
TUERK, C;GOLD, L
通讯作者：
GOLD, L