Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis

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.

Identification and Validation Model for Informative Liquid Biopsy-Based microRNA Biomarkers: Insights from Germ Cell Tumor In Vitro, In Vivo and Patient-Derived Data

“…Hence, it is fair to assume that the same cutoffs determined by the authors are not necessarily applicable in our workflow. Indeed, hemolysis did not show significant direct impact on the microRNA isolation procedure (denoted by ath-miR-159a) nor on the specific target assay miR-372a-3p, which contrasts with the findings of Myklebust and coworkers [43]. An effect on the normalizer miR-30b-5p levels was depicted; however, such effect was minor and seen mainly at the expense of cases with severe hemolysis, with visual scores of 4-5 ( Figure 8), which were absent in our study cohorts.…”

“…The value of this standardized pipeline relies on appropriate quality control and normalization [23,29]. However, some important issues remain, such as hemolysis content in blood samples, as it can interfere with the detection levels of certain microRNAs [42,43], as well as the choice of analysis of serum or plasma. However, the real impact of these matters on specific assays and the best way to approach them is still debatable.…”

MicroRNAs as Appropriate Discriminators in Non-Specific Alpha-Fetoprotein (AFP) Elevation in Testicular Germ Cell Tumor Patients

“…First, the lack of a pre-operative serum samples to confirm the detection of miR-371a-3p in the patient which enables us only to be able to indirectly conclude the high specificity of this biomarker under this condition based on our previous findings [15]. Another limitation is the use of the proper housekeeping gene from serum, as some newer studies also reported issues about pre-analytical influence of haemolysis on the concentration of the serum-based microRNAs [20,21]. In addition, we cannot rule out a lack of expression of this microRNA in the cancer tissue of our patient.…”

Non-Coding microRNAs as Novel Potential Tumor Markers in Testicular Cancer

“…As a further consequence, the determination of miR-371a-3p and miR-372-3p (which themselves are not affected by hemolysis) can be falsified, thus adversely affecting the diagnosis. Therefore, hemolysis and any kind of contamination of the samples should be avoided, or other miRNAs should be considered as endogenous calibrators and normalizers [85].…”