Chia‑Lin Yanga, Fu‑Ming Tsaib, Chuang‑Wei Chenc, Kuan‑Hong Hsiaoc, Jia‑Hui Chend, Woei‑Yau Kaoa, e*
aDivision of Hematology‑Oncology, Department of Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei, Taiwan; bDepartment of Research, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei, Taiwan; cDivision of Colon and Rectal Surgery, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei, Taiwan; dDivision of General Surgery, Department of Surgery, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei, Taiwan;
eSchool of Medicine, Tzu Chi University, Hualien, Taiwan
Open Access funded by Buddhist Compassion Relief Tzu Chi Foundation
Abstract
Objectives: Circulating microRNAs (miRNAs) have been discovered to play a novel role in intercellular communication and cancer biology. They are emerging candidates for noninvasive molecular biomarkers of cancer and other diseases. However, current translational researches have been limited by the lack of consensus on the optimal endogenous control of circulating miRNAs quantitation. In this study, we compared two promising miRNAs, miR‑1228 and miR‑16, as an endogenous control. The effects of normalizers on the relative quantification of circulating miR‑31 in plasma samples of colorectal cancer (CRC) were also assessed. Materials and Methods: The cel‑miR‑39 was a spiked‑in RNA used as an external control and added to plasma samples before RNA extraction. Quantitative real‑time polymerase chain reaction technology was used to analyze the expression levels of circulating miRNAs in plasma samples of 4 healthy controls and 14 CRC patients. The expression stability of the candidate controls was compared by Ct analysis and NormFinder algorithms. Results: There was no significant difference in expression level of miR‑16 and miR‑1228 between healthy control group and before or after therapy of CRC patient groups. The expression of miR‑1228 has smaller the range Ct values (28.25‑25.64) compared with those of miR‑16 (24.91‑20.34). The stability value of miR‑1228 (0.102) is lower than that of miR‑16 (0.350). The expression of miR‑1228 endogenous reference candidate has lower stability value and smaller the range Ct values compared with those in miR‑16. According to the range Ct values and stability value, miR‑1228 is better than miR‑16 as endogenous control in CRC patients. There are significant differences in circulating miR‑31 expression between healthy control and CRC patients when miR‑1228 was used to standardize miR‑31 expression. Conclusions: miR‑1228 is recommended as a better endogenous control in quantification of circulating miRNAs in CRC patients.
Keywords: Circulating microRNAs, Colorectal cancer, Quantification
