Digital PCR (dPCR) Quantification of miR-155-5p as a Potential Candidate for a Tissue Biomarker of Inflammation in Rabbits Infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV)

Abstract

MicroRNAs (miRNAs, miRs) are a group of small, 17-25 nucleotide, non-coding RNA sequences that, in their mature form, regulate gene expression at the post-transcriptional level. They participate in many physiological and pathological processes in both humans and animals. One such process is viral infection, in which miR-155 participates in innate and adaptive immune responses to a broad range of inflammatory mediators. Recently, the study of microRNA has become an interesting field of research as a potential candidate for biomarkers for various processes and disease. To use miRNAs as potential biomarkers of inflammation in viral diseases of animals and humans, it is necessary to improve their detection and quantification. In a previous study, using reverse transcription real-time quantitative PCR (RT-qPCR), we showed that the expression of ocu-miR-155-5p in liver tissue was significantly higher in rabbits infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV) compared to healthy rabbits. The results indicated a role for ocu-miR-155-5p in Lagovirus europaeus/RHDV infection and reflected hepatitis and the impairment/dysfunction of this organ during RHD. MiR-155-5p was, therefore, hypothesized as a potential candidate for a tissue biomarker of inflammation and examined in tissues in Lagovirus europaeus/RHDV infection by dPCR. The objective of the study is the absolute quantification of ocu-miR-155-5p in four tissues (liver, lung, kidney, and spleen) of rabbits infected with Lagovirus europaeus/RHDV by digital PCR, a robust technique for the precise and direct quantification of small amounts of nucleic acids, including miRNAs, without standard curves and external references. The average copy number/µL (copies/µL) of ocu-miRNA-155-5p in rabbits infected with Lagovirus europaeus GI.1a/Rossi in the liver tissue was 12.26 ± 0.14, that in the lung tissue was 48.90 ± 9.23, that in the kidney tissue was 16.92 ± 2.89, and that in the spleen was 25.10 ± 0.90. In contrast, in the tissues of healthy control rabbits, the average number of copies/µL of ocu-miRNA-155-5p was 5.07 ± 1.10 for the liver, 23.52 ± 2.77 for lungs, 8.10 ± 0.86 for kidneys, and 42.12 ± 3.68 for the spleen. The increased expression of ocu-miRNA-155-5p in infected rabbits was demonstrated in the liver (a fold-change of 2.4, p-value = 0.0003), lung (a fold-change of 2.1, p-value = 0.03), and kidneys (a fold-change of 2.1, p-value = 0.01), with a decrease in the spleen (a fold-change of 0.6, p-value = 0.002). In the study of Lagovirus europaeus/RHDV infection and in the context of viral infections, this is the first report that shows the potential use of dPCR for the sensitive and absolute quantification of microRNA-155-5p in tissues during viral infection. We think miR-155-5p may be a potential candidate for a tissue biomarker of inflammation with Lagovirus europaeus/RHDV infection. Our report presents a new path in discovering potential candidates for the tissue biomarkers of inflammation.

Keywords: Rabbit Hemorrhagic Disease Virus (RHDV); biomarker; digital PCR (dPCR); inflammation; microRNA-155 (miR-155); tissues.

Figure 1

Scheme of the digital PCR (dPCR) quantification of microRNA (miR-155-5p) in the tissues of rabbits infected with Lagovirus europaeus (L. europaeus)/Rabbit Hemorrhagic Disease Virus (RHDV). The research used the QIAcuity Digital PCR System, Qiagen, Hilden, Germany. (A) Sample preparation (dPCR reaction mix containing the target sequences miR-155-5p and background; see the Methods Section). (B) Partitioning (performed on a special 8.5k nanoplate; the sample is divided into many independent partitions such that each contains one or no target sequences of interest). (C) Amplification (PCR) (each partition acts as an individual PCR microreactor, amplifying the target sequences in each of them). (D) Detection (partitions that contain the amplified target miR-155-5p are detected by fluorescence). The ratio of positive partitions (presence of fluorescence) over the total number allows for the determination of the concentration of the target in the sample. (E) Data analysis (data output from dPCR assay to the absolute quantification of miR-155-5p in rabbit tissues infected with L. europaeus). One-dimensional scatterplots of event number–droplets vs. fluorescence amplitude for an ideal assay with a clear separation of positive (blue) and negative (grey) droplets.

Figure 2

Absolute quantification of ocu-miR-155-5p by dPCR. Liver (A), lung (B), kidney (C), and spleen (D). Data are shown as the mean ± standard error (SE), and p ≤ 0.05 was considered significant.