The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing

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Abstract
In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R2 = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future.

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