Sai Mun Leong1,2, Karen Mei-Ling Tan3, Hui Wen Chua2, Mo-Chao Huang3, Wai Chye Cheong4, Mo-Huang Li4, Steven Tucker5, Evelyn Siew-Chuan Koay1,2. 1. Department of Laboratory Medicine, NUH, Singapore, Singapore; patv29@nus.edu.sg evelyn_koay@nuhs.edu.sg. 2. Department of Pathology, Yong Loo Lin School of Medicine, NUS, Singapore, Singapore. 3. Department of Laboratory Medicine, NUH, Singapore, Singapore. 4. CellSievo Pte Ltd., Singapore, Singapore. 5. Tucker Medical, Novena Specialist Center, Singapore, Singapore.
Abstract
BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. METHODS: Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA)® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. RESULTS: We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. CONCLUSIONS: Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses.
BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. METHODS: Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancerpatient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA)® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. RESULTS: We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancerpatients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. CONCLUSIONS: Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses.