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Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate)
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-10-16 00:00:00 , DOI: 10.1021/acs.analchem.8b02338
Sangamithirai Subramanian Parimalam 1, 2 , Yusuke Oguchi 1, 3 , Mahmoud N. Abdelmoez 1, 2 , Arata Tsuchida 1, 2 , Yuka Ozaki 1 , Ryuji Yokokawa 2 , Hidetoshi Kotera 2 , Hirofumi Shintaku 1
Affiliation  

We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.

中文翻译:

双硫双(丙酸琥珀酰亚胺酯)固定的单细胞的电裂解和RNA提取

我们提出了一种利用可逆交联剂二硫代双(琥珀酰亚胺丙酸酯)(DSP)从单个固定细胞中进行细胞裂解和RNA提取的微流方法。我们的微流体系统在流体动力阱处捕获单个DSP固定的细胞,将芯片上的DSP分子与二硫苏糖醇反向交联,通过电场裂解质膜,并通过等速电泳辅助核酸提取来提取细胞质RNA。所有片上过程均在不到5分钟的时间内完成。我们证明了使用K562白血病细胞的方法,并通过逆转录定量聚合酶链反应确定了RNA提取的性能。我们还展示了我们的方法与单细胞RNA测序的整合。
更新日期:2018-10-16
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