Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification

Abstract

Alterations in microRNA (miRNA) expression patterns have been associated with a number of human diseases. Accurate quantitation of miRNA levels is important for their use as biomarkers and in determining their functions. Although the issue of proper miRNA detection was solved with the introduction of standard reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) assays, numerous issues with the selection of appropriate internal control genes remain. U6 (RNU6‑1) snRNA, the most commonly used internal control gene in miRNA RT‑qPCR assays, was shown to be unstable in clinical samples, particularly cancer tissues. Identification of the distribution of U6 in different tissues is the premise of more accurate quantification of miRNAs. However, the distribution of U6 in human carcinoma tissues and corresponding normal tissues is unknown. In the present study, U6 levels were significantly higher in human breast carcinoma tissues compared with the corresponding normal tissues by RT‑qPCR. In the carcinoma or corresponding adjacent normal tissues, the expression levels of U6 in epithelial cells were higher than those in the mesenchymal cells. Furthermore, the expression levels of U6 in the carcinoma tissues of the liver and intrahepatic bile ducts were higher than those in the adjacent normal tissues. These results suggest that the expression and distribution of U6 exhibits a high degree of variability among several types of human cells. Therefore, caution is required when selecting U6 as an internal control gene for evaluating expression profiles of miRNAs in patients with carcinoma, particularly carcinoma of the liver and intrahepatic bile ducts.

Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity

“…For example, it has previously been shown that U6 and rRNA5s were overexpressed in serum from breast cancer patients’ respect to healthy women’ serum23. Furthermore, U6 expression levels in liver and breast cancer tissues were higher than those expressed in adjacent normal tissues24. These studies support the notion that the expression and distribution of U6 and rRNA5s exhibits a high degree of variability among several types of human cells.…”

Identification of stably expressed reference small non‐coding RNAs for microRNA quantification in high‐grade serous ovarian carcinoma tissues

“…Notably, SNORD48 was already reported among the most stably expressed sncRNAs in endometrial cancer and renal cell carcinoma tissues . Conversely, U6, the most commonly reported normalization sncRNAs for miRNA expression studies, did not fulfil the criteria of constant expression between our cohorts of normal and tumour samples, as already described for other malignancies .…”

“…Actually, most of the investigations report arbitrarily chosen endogenous controls, including miRNA, snRNA and snoRNA, without any experimental validation of their stability. In addition, the large majority of these studies report U6 (alias RNU6‐1) as an endogenous reference, although its use is still controversial, as a growing body of evidence demonstrate its high expression instability across normal and tumour tissues .…”

Long non‐coding RNA linc01433 promotes migration and invasion in non‐small cell lung cancer

“…Our results revealed a higher level of linc01433 expression in three of the NSCLC cell lines (A549, H358, and PC9) than in BEAS‐2B, while no significant difference was identified in H520 or H1299 ( P < 0.01) (Fig a). Furthermore, we determined the localization of linc01433 in cells; GAPDH and small nuclear RNA U6 were used as internal controls of the cytoplasm and nucleus, respectively . The results of qRT‐PCR analysis of linc01433 expression levels in the nuclear/cytoplasmic fractions in A549 and H358 cells are presented in Figure b,c.…”