Detection of piRNA‐54265 in human serum: evidence and significance

Dear Editor, An increasing number of studies have shown that PIWIinteracting RNAs (piRNAs) are aberrantly expressed in many types of human cancer and involved in regulating the malignant progression, piRNAs would be expected to serve as molecular biomarkers for cancer screening and early diagnosis. In a previous study, we have identified the presence of piRNA-54265 in human serum [1]. Further research has demonstrated that it is a promising and specific blood molecular marker for colorectal cancer (CRC) [2]. In these studies, we have developed a stem-loop primer reverse transcription-quantitative PCR (RT-qPCR) approach to determine the piRNA-54265 levels, which included the reverse transcription of piRNA-54265 to cDNA using a stem-loop primer and the quantitative detection of cDNA by PCR with the primers and probe specific for piR-54265 [2]. However, a recent report suggested that piR-54265 we detected in serumcould be a full-length (72 nt) small nucleolar RNA-SNORD57 [3]. To clarify this important issue, we performed further experiments to validate our method of piRNA detection and verify the identity of piR-54265 in human serum samples. The new results further confirmed our previous findings that the RNA detected in human serum was indeed mature piR-54265, but not SNORD57, based on the following evidences. The stem-loop primer RT-PCR method we developed for detecting piR-54265 is shown in Figure 1A. As the name implies, the stem-loop reverse transcription primer includes two segments completely complementary with each other to form a stem loop, as indicated by the black boxes. Besides, our primer contains 9 bases complementary with piR-54265 at 3’ end (highlighted in yellow) and the rest bases of piR-54265 from its 5’ end to form