Abstract
Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
Highlights
Hemolysis could be detected at approximately 0.06% (v/v) Red blood cells (RBCs) in blood, but the hemolysis did not appear pronounced until 0.25% (v/v)
Quantification by real-time quantitative PCR (RT-qPCR) of the miRNAs proposed as endogenous controls in analysis of microRNAs 371a-372 in serum showed that the concentration of miR-191-5p and miR30b-5p did increase in serum when hemolysis occurs, but the increase in miR-93-5p and miR-20a-5p were more pronounced (Figure 2)
According to the Cq-values shown in Figure 2, the levels of miR-93-5p and miR-20a-5p increased in serum from the lowest amount of 0.008% added RBCs
Summary
MicroRNAs of the miR-371-373 cluster, which are produced in embryonal tumors, have been proposed as sensitive biomarkers for testicular germ cell tumors (GCT), both in primary cases and recurrences (Suh et al, 2004; Belge et al, 2012; Dieckmann et al, 2012; Gillis et al, 2013; Syring et al, 2015; Murray et al, 2016; Anheuser et al, 2017; Terbuch et al, 2018; Dieckmann et al, 2019). miR-371a-3p, which has the highest sensitivity and specificity of these miRNAs, has been shown to be expressed in both seminomas and non-seminomas, except pure teratomas (Dieckmann et al, 2016; Vilela-Salgueiro et al, 2018). Dieckmann et al (2017) reported miR-371a-3p to detect GCT with sensitivity and specificity of 88.7 and 93.4%, respectively. Free, circulating microRNAs in serum and plasma as biomarkers is warranted as it is a simple, non-invasive technique with little discomfort for the patient. The minute amounts of specific microRNAs in serum and plasma need very sensitive methods for detection. Reference microRNAs must be expressed at similar levels throughout all samples tested, both in serum or plasma from individuals with the disease in question and in healthy controls. Suitable reference microRNAs are often expressed at high levels in blood cells. We show how hemolysis affects four microRNAs used as reference genes in the quantitative analysis of miR-371a-3p and miR-372-3p. MiR-93-5p, miR-20a-5p, and miR-30b-5p have been used as reference genes in analysis of the miR-371-373 cluster in serum (Gillis et al, 2013; Murray et al, 2016; Dieckmann et al, 2017). We demonstrate how hemolysis will affect the calculated relative quantity (RQ) of miR-371a -3p and miR-372-3p in patient samples
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