Considerations and Suggestions for the Reliable Analysis of miRNA in Plasma Using qRT-PCR

Abstract

MicroRNAs (miRNAs) are promising molecules that can regulate gene expression, and their expression level and type have been associated with early diagnosis, targeted therapy, and prognosis of various diseases. Therefore, analysis of miRNA in the plasma or serum is useful for the discovery of biomarkers and the diagnosis of implicated diseases to achieve potentially unprecedented progress in early treatment. Numerous methods to improve sensitivity have recently been proposed and confirmed to be valuable in miRNA detection. Specifically, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and common method for sensitive and specific analysis of miRNA from biological fluids, such as plasma or serum. Despite this, the application of qRT-PCR is limited, as it can be affected by various contaminants. Therefore, extraction studies have been frequently conducted to maximize the extracted miRNA amount while simultaneously minimizing contaminants. Moreover, studies have evaluated extraction efficiency and normalization of the extracted sample. However, variability in results among laboratories still exists. In this review, we aimed to summarize the factors influencing the qualification and quantification of miRNAs in the plasma using qRT-PCR. Factors influencing reliable analysis of miRNA using qRT-PCR are described in detail. Additionally, we aimed to describe the importance of evaluating extraction and normalization for reliable miRNA analysis and to explore how miRNA detection accuracy, especially from plasma, can be improved.

Keywords: amplification efficiency; miRNA; plasma; qRT-PCR.

Figure 1

The number of PubMed search results regarding articles reporting on analyses of circulating miRNAs.

Figure 2

Methods for endogenous miRNA analysis in plasma or serum compared in terms of the proportions of articles reporting their use among PubMed search results from the past 10 years.

Figure 3

Effects of normalization by different reference gene methods on the expression levels of miRNAs from plasma samples of stable coronary artery disease patients and healthy controls (n = 5 in each group). For each group, 2 µL of exogenous cel-miR-39 was spiked into 300 µL plasma. The levels of cel-miR-39 were assessed by qRT-PCR and were normalized to miR-16, miR-6090, miR-4516, miR-484, and RNU6 [62].

Figure 4

Suggested flowchart for qRT-PCR analysis of plasma miRNA.