Abstract
We have developed an optimized protocol for plasma targeted mRNA sequencing in our previous study. Here, we performed plasma targeted mRNA sequencing for 40 colorectal adenoma patients and 39 colonoscopy-proven normal controls in order to find potential circulating mRNA markers for colorectal adenoma. Results showed that GSK3A and RHOA were differential expressed genes identified by a cut-off of fold change >2 and adjusted P value < 0.05. More detailed analysis showed that the expression of both GSK3A (0.01-fold with adjusted P < 1 × 10−6) and RHOA (0.35-fold with adjusted P < 0.01) in adenoma patients was significantly lower than those in normal healthy subjects. Based on the enrichment analysis of biological process for potential markers, we found that the regulation of programmed cell death (GO: 0043067; GO: 0043069), regulation of cell death (GO: 0010941; GO: 0060548) and cell differentiation (GO: 0021861) were the main processes involved in adenoma formation. In summary, this study is a cutting-edge research on the detection of plasma mRNA in colorectal adenoma patients and normal healthy subjects.
Highlights
The similarity of samples was visualized by the clustering based on Euclidean distance (Fig. 2c) and principal components analysis (PCA) (Fig. 2d)
Normal plasma sample and adenoma plasma sample had different expression patterns compared with other samples, which was demonstrated by the PCA result
We focus on colorectal adenoma instead of CRC because not all colorectal adenoma will develop into CRC
Summary
Forty colorectal adenoma patients and 39 normal healthy controls were recruited from the Department of Surgery, Queen Elizabeth Hospital, Hong Kong. Those normal controls were colonoscopy confirmed to be healthy. Anti-coagulated blood was collected by K3 EDTA tubes (Greiner Bio-one, Austria) and centrifuged for 1,600 g, 10 minutes at 4 °C. The clear upper layer www.nature.com/scientificreports plasma was collected and re-centrifuged for 16,000 g, 10 minutes at 4 °C to remove residual cell pellet. 2.5 ml cell free plasma was collected and preserved by 2 ml Trizol Reagent (Thermo Fisher Scientific, USA) before storage at −80 °C. Blood processing was performed within 4 hours after blood draw
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