RNA capture pin technology: investigating long-term stability and mRNA purification specificity of oligonucleotide immobilization on gold and streptavidin surfaces
RNA capture pin technology: investigating long-term stability and mRNA purification specificity of oligonucleotide immobilization on gold and streptavidin surfaces
复制标题
DOI：
10.1007/s00216-023-04882-6
复制
发表时间：
2023-07-29
影响因子：
4.3
通讯作者：
Nestorova, Gergana G.
中科院分区：
文献类型：
--
作者：
Gaines, Deriesha;Brodsky, Elia;Nestorova, Gergana G.
推进生物医学研究需要开发用于快速提取核酸的尖端技术。我们表征了RNA捕获针（RCP）工具，其对样品无破坏性，并且能够快速纯化和富集mRNA用于随后的遗传分析。该技术的核心是用dT 15捕获序列功能化的针（200 μ m × 3cm），所述dT 15捕获序列在插入样本的2分钟内与mRNA杂交。研究了两种将寡核苷酸固定在RCP表面的方法：金-硫醇法和生物素-链霉亲和素法。使用萝卜植物评估RCP的RNA捕获效率。平均逆转录定量聚合酶链反应（RT-qPCR）循环扩增值分别为19.93和24.84的金和链霉亲和素包被针。使用Agilent 2100生物分析仪测量探针表面上存在的RNA的量。进行RNA测序以确定RNA捕获针的mRNA选择性。基因读段计数分析证实，通过镀金RCP纯化的RNA含有70%的信使RNA，10%的核糖体RNA和20%的非编码RNA。在4个月内评估dT 15寡核苷酸与RCP表面之间键合的长期稳定性。在4 ℃下储存2周后，观察到链霉亲和素包被的RCP的dT 15表面覆盖率显著降低。金-硫醇RNA捕获针在储存4个月后显示出40%的寡核苷酸的留存率。
Advancing biomedical studies necessitates the development of cutting-edge technologies for the rapid extraction of nucleic acid. We characterized an RNA capture pin (RCP) tool that is non-destructive to the sample and enables rapid purification and enrichment of mRNA for subsequent genetic analysis. At the core of this technology is a pin (200 & mu;m x 3 cm) functionalized with dT15 capture sequences that hybridize to mRNA within 2 min of insertion in the specimen. Two methods for immobilizing the oligos on the surface of the RCPs were investigated: gold-thiol and biotin-streptavidin. The RNA capture efficiency of the RCPs was assessed using a radish plant. The average reverse transcription-quantitative polymerase chain reaction (RT-qPCR) cycle amplification values were 19.93 and 24.84 for gold- and streptavidin-coated pins, respectively. The amount of RNA present on the surface of the probes was measured using the Agilent 2100 Bioanalyzer. RNA sequencing was performed to determine the mRNA selectivity of the RNA capture pin. Gene read count analysis confirmed that the RNA purified via the gold-plated RCPs contained 70% messenger RNA, 10% ribosomal RNA, and 20% non-coding RNA. The long-term stability of the bond between the dT15 oligos and the surface of the RCPs was assessed over 4 months. A significant decrease in the dT15 surface coverage of the streptavidin-coated RCPs was observed after 2 weeks of storage at 4 & DEG;C. The gold-thiol RNA capture pins exhibited a retention rate of 40% of the oligos after 4 months of storage.