U6 is not a suitable endogenous control for the quantification of circulating microRNAs

https://doi.org/10.1016/j.bbrc.2014.10.064Get rights and content

Highlights

  • Stability of U6 expression was less than miR-16 in different populations’ serum.

  • U6 expression decreased after cycles of freezing and thawing.

  • MiR-16 and miR-24 were stable relatively after cycles of freezing and thawing.

  • U6 is not a suitable endogenous control of circulating microRNAs.

Abstract

Recently, microRNAs have been detected in serum and plasma, and circulating microRNA (miRNA) profiles have now been associated with many diseases such as cancers and heart disease, as well as altered physiological states. Because of their stability and disease resistance, circulation miRNAs appear to be an ideal material for biomarkers of diseases and physiological states in blood. However, the lack of a suitable internal reference gene (internal reference miRNA) has hampered research and application of circulating miRNAs. Currently, U6 and miR-16 are the most common endogenous controls in the research of miRNAs in tissues and cells. We performed microarray-based serum miRNA profiling on the serum of 20 nasopharyngeal carcinoma patients and 20 controls to detect the expressions of U6 and miRNAs. Profiling was followed by real-time quantitative Polymerase Chain Reaction (qPCR) in 80 patients (20 each with gastric cancer, nasopharyngeal carcinoma, colorectal cancer, and breast cancer) and 30 non-cancerous controls. qPCR was also performed to detect miRNAs in serum with repeated freezing and thawing. The results of microarray showed that with the exception of U6, Ct values of miR-16, miR-24, miR-142-3p, miR-19b and miR-192 in serum samples of nasopharyngeal carcinoma were greater than control samples. The results of 110 cases showed large fluctuations in U6 expression. The difference between the greatest and the least levels of expression was 3.29 for delta Ct values, and 1.23 for miR-16. The expressions of U6, miR-16 and miR-24 in serum subjected to different freeze–thaw cycles showed that U6 expression gradually decreased after 1, 2, and 4 cycles of freezing and thawing, while the expression of miR-16 and miR-24 remained relatively stable. Collectively, our results suggested that U6 is unsuitable as an internal reference gene in the research of circulating miRNAs.

Introduction

MicroRNAs (miRNAs) play a key role in the development of many diseases, which negatively regulate the expression of protein-coding genes at the post-transcriptional level [1], [2]. Particularly in tumors, miRNAs play a role similar to that of oncogenes or tumor suppressor genes [1], [3]. In 2008, several research groups have discovered miRNA in human serum and plasma [4], [5]. These circulating miRNAs are very stable in the blood. Healthy individuals have a consistent level of circulating miRNAs expression, but the levels of circulating miRNAs in serum will significantly change in different diseases [5]. Therefore, circulating miRNAs can become potential biomarkers for the diagnosis, monitoring, and prognosis of diseases.

Although research on circulating miRNAs as biomarkers of disease is on the rise, researchers are encountering a practical problem, the lack of a recognized and reliable reference gene (housekeeping gene), which makes comparisons difficult between different samples and complicates the search for biomarkers in circulating miRNAs. Currently, researchers use U6 and miR-16 for internal reference in tissue and cell miRNAs studies. Researchers use the following ways to solve the problem of internal reference in circulating miRNAs detection: (a) According to reports in the literature , researchers often consider stable RNA expression as an internal reference of circulating miRNAs detection: e.g. U1 [6], U6 [7], [8], [9], U43 [6], U44 [10], [11], U48 [10], [12], [13], miR-16 [14], [15], [16], [17], [18], [19], miR-19b [4], miR-24 [4], [14], miR-30e [20], miR-142-3p [10], miR-192 [21], miR-638 [22], let-7a [14], 5S [23], [24], 18S [25], etc. However, failure to undergo rigorous experimental verification has led to disagreement among researchers [26], thus some results have been questioned. Especially, there are much controversy about whether U6 and miR-16 are good internal references [5], [7], [8], [9], [14], [15], [16], [17], [18], [19]. (b) Added the same amount of exogenous miRNAs (sample species does not contain) as internal reference into serum or plasma samples [27], [28], [29], [30], [31], [32]. This method can eliminate some deviations of the experimental process and make the results reliable. But it also causes the experimental procedure cumbersome and clinical applications more inconvenient. (c) Use concentration of total RNA samples as the reference index [5]. In this method, the accuracy of RNA concentration measurement has strict requirements. But the low circulating miRNAs levels are difficult to measure. In addition, concentration of total RNA may not give a true reflection of the amount of miRNA samples. (d) some researchers directly compare the volume obtained by equal amounts of serum/plasma sample [33], [34], [35]. Because the internal reference is not set, the deviation between the samples cannot be corrected, reducing the credibility of the results [36]. Taken together, there is still no recognized and reliable reference gene in circulating miRNAs detection, which seriously interferes with investigators and limits the development of circulating miRNA study. Therefore, screening current miRNAs used as internal references and finding reliable internal references of circulating miRNAs is urgently needed.

In this study, we screened and compared the expression level of some miRNAs used as internal controls, and found that U6 expression is not stable in serum. It may be not suitable as an internal reference gene in the study of circulating miRNAs.

Section snippets

Clinical samples

Following ethical approval and written informed consent, serum samples were collected from 150 individuals, including 100 patients (40 nasopharyngeal cancer, 20 gastric cancer, 20 colorectal cancer and 20 breast cancer, confirmed by pathology) and 50 non-cancerous volunteers. Among them, 80 patients and 30 non-cancerous volunteers were used for the validation (Table 1). All the patients had been diagnosed pathologically and their relevant demographic and clinical pathological details were

Microarray analysis of serum miRNA

The serum miRNA profile in NPC patients or non-cancerous controls was obtained by using TaqMan Low-Density Array. To screen endogenous control, six miRNAs was chosen, including U6, miR-16, miR-24, miR-142-3p, miR-19b and miR-192, which were reported in the literature as having stable expression in serum/plasma. Comparative analysis of microarray data was performed. The Ct values of these miRNAs in serum samples of nasopharyngeal carcinoma were greater than non-cancer serum samples, with the

Discussions

Theoretically, serum and plasma have the same composition except for blood coagulation factors. Some research groups have found that miRNA expression in serum and plasma are consistent [5]. Thus, the internal reference of serum and plasma are likely to be in common. In this study, we used serum for related research.

We analyzed the miRNAs reported to have stable expression in serum and which could be used as internal references. We found that with the exception of U6, the values for miR-16,

Acknowledgments

This work is supported by the National Natural Science Foundation of China (81101643); The Foundation of the Construct Program of the Key Discipline in Hunan Province of China ((2011)76); The Foundation of the Department of Health of Hunan Province (B2012-044).

The authors wish to thank Dr. Wang Chun of Southern Illinois University for modification to language of this article.

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    These authors contributed equally to this work.

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