Dataset Information

Challenges and standardization of microRNA profiling in serum and cerebrospinal fluid in dogs suffering from non-infectious inflammatory CNS disease.

ABSTRACT: Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demonstration of inflammatory changes in the cerebrospinal fluid (CSF). However, less-invasive methods and peripheral biomarkers are desired. Changes in circulating microRNA (miRNA), which are short non-coding regulatory RNAs, may serve as biomarkers of disease. The aim of this pilot study was to investigate selected miRNAs in serum and CSF, hypothesizing that the levels of specific miRNAs in serum correlate with their presence in CSF, and that changes in serum miRNAs levels may reflect CNS disease. We profiled serum and CSF samples using quantitative real-time PCR (qPCR) searching for selected and previously profiled miRNAs in serum (let-7a, let-7c, miR-15b, miR-16, miR-21, miR-23a, miR-24, miR-26a, miR-146a, miR-155, miR-181c and miR-221-3p) and in CSF (let-7c, miR-16, miR-21, miR-24, miR-146a, miR-155, miR-181c and miR-221-3p) from 13 dogs with NII CNS disease and six control dogs. We demonstrated the presence of several miRNAs in CSF (let-7c and miR-21 dominating) and serum (miR-23a and miR-21 dominating). However, we generally failed to reproduce consistent results in CSF samples due to several reasons: unacceptable PCR efficiency, a wide variation between cDNA replicates and/or no-amplification in qPCR suggesting very low levels of the investigated miRNAs in canine CSF. Serum samples performed better, and 10 miRNAs qPCR assays were qualified for analysis. We were nevertheless unable to detect a difference in the expression of miRNA levels between cases and controls. Moreover, we could not confirm the results of recent miRNA investigations of canine CNS diseases. We believe that these disagreements highlight the significant effect of methodological/analytical variation, rather than the incapacity of circulating miRNAs as biomarkers of CNS disease. A secondary aim was therefore to communicate methodological challenges in our study and to suggest recommendations for circulating miRNA profiling, including pre-, post- and analytical methods based on our experience, in order to reach reproducible and comparable results in veterinary miRNA research.

SUBMITTER: Cirera S

PROVIDER: S-EPMC6889416 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Challenges and standardization of microRNA profiling in serum and cerebrospinal fluid in dogs suffering from non-infectious inflammatory CNS disease.

Cirera Susanna S Andersen-Ranberg Emilie Ulrikka EU Langkilde Sille S Aaquist Maria M Gredal Hanne H

Acta veterinaria Scandinavica 20191203 1

Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demonstration of inflammatory changes in the cerebrospinal fluid (CSF). However, less-invasive methods and peripheral biomarkers are desired. Changes in circulating microRNA (miRNA), which are short non-coding regulatory RNAs, may serve as biomarkers of disease. The aim of this pilot study was to investigate selected miRNAs in serum and CSF, hypothesizing that the levels of specific miRNAs in ...[more]

PMID: 31796072

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Project description:BackgroundDistinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique.MethodsIn this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease.ResultsBlood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days).ConclusionsThese results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Project description:IntroductionNon-infectious inflammatory diseases of the central nervous system in dogs, such as steroid responsive meningitis-arteritis (SRMA) and meningoencephalitis of unknown origin (MUO), represent a common clinical challenge that needs extensive and multimodal work-up to reach a presumptive diagnosis. Both diseases are presumably caused by dysregulations of the immune system, but further research is needed in order to understand the molecular mechanisms behind each disease and to optimize treatment.MethodsBy next-generation sequencing and subsequent quantitative real-time PCR (qPCR) verification, we designed a prospective case-control pilot study to analyze the small RNA profiles of cerebrospinal fluid from dogs suffering from MUO (N = 5), dogs suffering from SRMA (N = 8), and healthy dogs (N = 5) presented for elective euthanasia used as the Control group.ResultsOur results showed an overall enrichment in Y-RNA fragments across all samples, followed by microRNAs (miRNAs) and ribosomal RNAs as the major findings. Additional traces of short RNA reads mapped to long non-coding RNAs and protein-coding genes were also found. From the detected canine miRNAs, miR-21, miR-486, miR-148a, miR-99a, miR-191 and miR-92a were among the most abundant. Dogs with SRMA showed higher differences in miRNA abundance than dogs with MUO when compared to healthy dogs, and miR-142-3p was consistently detected as differentially upregulated in both diseases, although at a low concentration. Moreover, miR-405-5p and miR-503-5p showed different profiles between SRMA and MUO dogs. Subsequent qPCR analyses confirmed miR-142-5p, miR-191-5p and miR-92a-3p as significantly upregulated miRNAs in dogs with SRMA and/or MUO.DiscussionCerebrospinal fluid is a challenging biological material to use for profiling miRNAs due to the low content of circulating RNAs. Despite this, we could confirm several miRNAs being differentially abundant when comparing healthy dogs and dogs with MUO and SRMA, respectively. The results of this study indicate a potential role of miRNAs in the underlying molecular mechanisms of these diseases and establish the basis for further studies.

Dataset Information

Challenges and standardization of microRNA profiling in serum and cerebrospinal fluid in dogs suffering from non-infectious inflammatory CNS disease.

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Challenges and standardization of microRNA profiling in serum and cerebrospinal fluid in dogs suffering from non-infectious inflammatory CNS disease.

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