Citation: PANG Shu-Yun, XU Shu, XU Zhao-Luo, CHU Ya-Nan, MA Xue-Ping, WU Hai-Ping, ZOU Bing-Jie, ZHOU Guo-Hua. Quality Evaluation of Capture and Single-cell Isolation of Circulating Tumor Cell Clusters for Single-cell Ribonucleic Acid Analysis[J]. Chinese Journal of Analytical Chemistry, ;2023, 51(4): 513-520. doi: 10.19756/j.issn.0253-3820.221526 shu

Quality Evaluation of Capture and Single-cell Isolation of Circulating Tumor Cell Clusters for Single-cell Ribonucleic Acid Analysis

PANG Shu-Yun ¹ ,
XU Shu ⁵ ,
XU Zhao-Luo ¹ ,
CHU Ya-Nan ¹ ,
MA Xue-Ping ¹ ,
WU Hai-Ping ^1,3 ,
ZOU Bing-Jie ^1,3,4,, ,
ZHOU Guo-Hua ^1,2,3,,

1.
Department of Clinical Pharmacy, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China;
2.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China;
3.
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China;
4.
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China;
5.
School of Basic Medical Science and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, China

Corresponding author: ZOU Bing-Jie, ZHOU Guo-Hua,
Received Date: 25 October 2022
Revised Date: 28 December 2022

Fund Project: Supported by the National Natural Science Foundation of China (No. 61871403).

Single-cell ribonucleic acid (RNA) analysis is necessary to investigate the biological effects of circulating tumor cell clusters (CTC clusters). To achieve the most efficient results in single-cell analysis of CTC clusters, the integrity of the CTC cluster and the high quality of the intracellular RNA should be maintained after the capture and single-cell isolation. So, three house gene was quantitated in single cells for RNA quality control by single-cell RNA sequencing method, and the CTC cluster's lateral diameter for evaluating the CTC cluster's integrity were used to evaluate the quality of CTC cluster capture and single-cell isolation process. Trypsin-EDTA and non-enzymatic cell dissociation solution (NECDS) were used to isolate single cells in CTC clusters, followed by the investigation of intracellular RNA quality. The CTC cluster recovery of negative enrichment, positive enrichment and membrane filtration, and single-cell RNA integrity after enrichment were investigated. The results indicated that the NECDS isolated single cells from CTC clusters rapidly without affecting the intracellular RNA. Besides, it was found that rotational incubation and treatments of red blood cell lysate disrupted the integrity of CTC clusters, therefore, these operations were not the best way for single-cell RNA analysis of CTC clusters. The positive enrichment with simplified process achieved a high recovery without affecting the quality of intracellular RNA. Single-cell RNA analysis of CTC clusters required gentle capture and single-cell isolation process. The present study here provided an efficient sample preparation strategy for single-cell analysis of CTC clusters.
- Circulating tumor cell clusters,
- Single-cell ribonucleic acid analysis,
- Single-cell isolation,
- Cluster capture
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