Melissa A. McAlexander

Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: Limited evidence for general uptake of dietary plant xenomiRs

Evidence that exogenous dietary miRNAs enter the bloodstream and tissues of ingesting animals has been accompanied by an indication that at least one plant miRNA, miR168, participates in “cross-kingdom” regulation of a mammalian transcript. If confirmed, these findings would support investigation of miRNA-based dietary interventions in disease. Here, blood was obtained pre- and post-prandially (1, 4, 12 h) from pigtailed macaques that received a miRNA-rich plant-based substance. Plant and endogenous miRNAs were measured by RT-qPCR. Although low-level amplification was observed for some plant miRNA assays, amplification was variable and possibly non-specific, as suggested by droplet digital PCR. A consistent response to dietary intake was not observed. While our results do not support general and consistent uptake of dietary plant miRNAs, additional studies are needed to establish whether or not plant or animal xenomiRs are transferred across the gut in sufficient quantity to regulate endogenous genes.

Comparison of Methods for miRNA Extraction from Plasma and Quantitative Recovery of RNA from Cerebrospinal Fluid

Interest in extracellular RNA (exRNA) has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific exRNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Toward these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and quantitative recovery of RNA is observed from increasing volumes of cerebrospinal fluid.

Association of BRAFV600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers

BACKGROUND Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM). METHODS Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC. RESULTS Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM. CONCLUSION In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.

Dietary flaxseed modulates the miRNA profile in irradiated and non-irradiated murine lungs: A novel mechanism of tissue radioprotection by flaxseed

Introduction Dietary flaxseed (FS) displays antioxidant and anti-inflammatory properties in preclinical models of lung disease including radiation-induced pneumonopathy, however the mechanisms of lung radioprotection are incompletely understood. MicroRNAs (miRNAs) are short oligonucleotides that act as important posttranscriptional regulators of diverse networks including inflammatory response networks. Responses of miRNA profiles to diet and radiation exposure have been reported, but the potential contribution of miRNAs to diet-related radioprotection has never been tested. Methods In this exploratory pilot study, mice were fed 10% FS or a 0% FS isocaloric control diet and exposed to a single-fraction 13.5 Gy thoracic X-ray radiation treatment (XRT). Lung RNA was extracted 48 h post-XRT and small RNAs profiled by OpenArray. Results FS significantly modulated expression of multiple miRNAs, including 7 with P < 0.001. miR-150 was downregulated approximately 2.9-fold in the FS groups and is disproportionately integrated into immune response-related networks. Although few miRNAs were significantly changed by radiation, interaction between diet and radiation was observed. For example, miR-29c was greatly downregulated in the FS/Control group (10- to 50-fold) but slightly upregulated in the FS/radiation group. Compared with FS/control, the FS/radiation group experienced a 50% decrease of the p53-responsive miR-34a, which regulates senescence- and apoptosis-related factors. Conclusions FS induced significant changes in lung miRNA profile suggesting that modulation of small RNA by dietary supplements may represent a novel strategy to prevent adverse side-effects of thoracic radiotherapy. This pilot study provides insight into a potential mechanism of flaxseed’s radioprotection and provides a useful model-system to further explore and optimize such small RNA-based therapies.

SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load.

Restriction of HIV-1 in myeloid-lineage cells is attributed in part to the nucleotidase activity of the SAM-domain and HD-domain containing protein (SAMHD1), which depletes free nucleotides, blocking reverse transcription. In the same cells, the Vpx protein of HIV-2 and most SIVs counteracts SAMHD1. Both Type I and II interferons may stimulate SAMHD1 transcription. The contributions of SAMHD1 to retroviral restriction in the central nervous system (CNS) have been the subject of limited study. We hypothesized that SAMHD1 would respond to interferon in the SIV-infected CNS but would not control virus due to SIV Vpx. Accordingly, we investigated SAMHD1 transcript abundance and association with the Type I interferon response in an SIV model. SAMHD1 transcript levels were IFN responsive, increasing during acute phase infection and decreasing during a more quiescent phase, but generally remaining elevated at all post-infection time points. In vitro, SAMHD1 transcript was abundant in macaque astrocytes and further induced by Type I interferon, while IFN produced a weaker response in the more permissive environment of the macrophage. We cannot rule out a contribution of SAMHD1 to retroviral restriction in relatively non-permissive CNS cell types. We encourage additional research in this area, particularly in the context of HIV-1 infection.

OpenArray profiling reveals no differential modulation of miRNA by positive and negative CD4+ T cell immunoselection

Blood cell subsets contain unique constellations of microRNAs [1,2], short oligonucleotide effectors of post-transcriptional gene regulation that are important from differentiation to disease [3]. In addition to the promise of small RNA-based therapeutics, expression profiles of these molecules may be useful for diagnosis, prognosis, and monitoring of response to therapy of diseases that affect cells of the blood. Whereas many thousands of messenger RNA species may be present in a given cell type, only several hundred distinct miRNAs, at most, are found within a single cell type, permitting relatively facile measurement and analysis. The conservation and relative lack of diversity of canonical miRNA variants may also allow high-level multiplexing in high throughput sequencing [4], lowering the cost barrier to development of clinically useful assays. However, selection of specific cell populations could conceivably confound miRNA profiling. Positive selection could stimulate target cells, altering small RNA profiles and thus masking disease-specific information or compromising the use of cells in downstream assays. Negative or “untouched” selection might better preserve native miRNA profiles but achieve less than complete removal of non-target cells, resulting in lower purity. We conducted a study to identify any large fold differences in miRNAs of CD4+ T-isolated by positive and negative immunoselection. CD4+ T-cells were isolated from de-identified human blood (New York Blood Center) using negative or positive selection (Miltenyi Biotec, #130-096-533 or #130-045-101, following depletion of CD14+ cells with #130-50-201, respectively). Cell purity was determined by flow cytometry as greater than 97% for positively and ~95% for negatively selected cells. Total cellular RNA was isolated using the mirVana protocol, and RNA integrity was verified by Agilent BioAnalyzer (RIN>9 for all samples). For each sample, 100 ng RNA was reverse-transcribed, pre-amplified, and profiled using miRNA OpenArray (Life Technologies) per manufacturer’s protocol. Quantile normalization was done using R/Bioconductor. Raw and quantile normalized data are available on request and will also be uploaded to the Gene Expression Omnibus. Three additional normalizations were to the geometric mean of all miRNA with average Crt >30 (“global geometric mean”); to the geometric mean of RNU48, RNU44, and snRNA U6 Crts; and to miR-16. Approximately 110 medium- to high-abundance miRNAs were detected in all samples [defined as amplification earlier than a relative threshold cycle (Crt) of 30]. Hierarchical clustering of quantile-normalized data revealed little support for global expression changes induced by positive selection of CD4+ T-cells (Figure 1A). For individual miRNAs, changes were not statistically significant following multiple comparison corrections. This outcome applied as well when data were normalized to the geometric mean of all miRNA with average Crt >30 (“global geometric mean”), the geometric mean of RNU48, RNU44, and snRNA U6 Crts, or to miR-16 alone. Evidence for influence of selection method was strongest for miR-130b (Figure 1B). In global geometric mean-normalized data, miR-130b was 1.8- to 3.7-fold more abundant in positively selected cells, and nominal “significance” of p<0.05 by paired t-test was recorded for this and for miR-16-normalized data. For results normalized by quantiles but not for the other methods, miR-128a appeared to be slightly downregulated in all positively selected samples, also with a nominally significant p value (Figure 1B). Importantly, however, no miRNA was significantly changed when significance tests were corrected for multiple comparisons. Figure 1 (A) CD4+ T-cells were isolated from PBMC of three donors (1–3) with positive (+) or negative “untouched” (−) bead-based selection. OpenArray profiling data for miRNAs amplifying before relative threshold cycle (Crt) of ... Confirmatory quantitative stem-loop RT-PCR was done with Life Technologies assays and reagents as described previously [5]. These data were normalized to the average of small RNAs RNU44 and U6. miRs-128a and -130b were tested based on the array results, along with several miRNAs with previously reported modulation in response to stimulation of T-cells or T-cell subsets: miRs-21 [1,6–8], -26a [1,6–9], -342-3p [1,7,9], and let-7g [6]. miR-16 and small RNAs RNU44 and snRNA U6 were also assayed. Cq values were normalized to the average of RNU44 and snRNA U6. Although very slight apparent upregulation of miR-130b and downregulation of miR-128a was again observed, no tested RNA species was significantly differentially expressed (Figure 1C). To confirm that the cells we used were capable of miRNA differential regulation upon stimulation, and that our approach could detect changes in the levels of miRNA in these cells, we stimulated CD4+ T-cells or not with anti-CD3 and anti-CD28 antibodies [10]. miRNAs were assayed by stem-loop RT-qPCR as described above. There was no differential regulation of miRs-16 and -128a or RNU44 and U6. Levels of miRs-21 and -130b increased, as reported elsewhere [1,6–8], while miRs-26a, -342-3p, and let-7g were downmodulated (Figure 1D). These results confirm the findings of other investigators as regards stimulation of T-cells and suggest that our system and techniques were appropriate for detection of real changes in miRNA levels. We conclude that brief exposure to anti-CD4 antibodies during positive cell selection did not stimulate large fold changes in miRNA expression when compared with a matched negative selection method. These results may bolster confidence that the judicious use of positive immunoselection, which may result in higher purity of cell populations, does not immediately or drastically alter the miRNA profile of target cells. Several limitations should be considered when interpreting this study. First, because of the small number of samples, small (i.e., less than two-fold) but consistent fold changes may have been overlooked. Investigators concerned that small fold changes of specific miRNAs could affect results might wish to pursue similar but larger studies. Second, we did not assess the behavior of positively and negatively selected cells in subsequent in vitro studies. Third, other cell types, selected using different surface markers, could be more or less sensitive to selection. Nevertheless, our findings offer reassurance that positive selection methods—which are relatively inexpensive on a per-cell basis and may achieve greater purity of isolated cells—may not unduly influence miRNA profiles and may thus be appropriate for isolation of cells for development of clinical assays.

miRNAs and SAMHD1 regulation in vitro and in a model of HIV CNS disease

Pilakka-Kanthikeel et al. recently reported higher levels of the retroviral restriction factor sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) in astrocytes than in microglia, suggesting that SAMHD1 levels might explain in part the relatively refractory nature of astrocytes to retroviral replication. These findings are consistent with our studies of simian and human immunodeficiency virus infection of astrocytes and macrophages. Similarly, a role for two host microRNAs in post-transcriptional regulation of SAMHD1 agrees with our in vitro results and those of others. However, data from an animal model of HIV neurologic disorders may not be consistent with robust miRNA-mediated regulation of SAMHD1 in vivo.

A benchmark for microRNA quantification algorithms using the OpenArray platform

BackgroundSeveral techniques have been tailored to the quantification of microRNA expression, including hybridization arrays, quantitative PCR (qPCR), and high-throughput sequencing. Each of these has certain strengths and limitations depending both on the technology itself and the algorithm used to convert raw data into expression estimates. Reliable quantification of microRNA expression is challenging in part due to the relatively low abundance and short length of the miRNAs. While substantial research has been devoted to the development of methods to quantify mRNA expression, relatively little effort has been spent on microRNA expression.ResultsIn this work, we focus on the Life Technologies TaqMan OpenArrayⓇ system, a qPCR-based platform to measure microRNA expression. Several algorithms currently exist to estimate expression from the raw amplification data produced by qPCR-based technologies. To assess and compare the performance of these methods, we performed a set of dilution/mixture experiments to create a benchmark data set. We also developed a suite of statistical assessments that evaluate many different aspects of performance: accuracy, precision, titration response, number of complete features, limit of detection, and data quality. The benchmark data and software are freely available via two R/Bioconductor packages, miRcomp and miRcompData. Finally, we demonstrate use of our software by comparing two widely used algorithms and providing assessments for four other algorithms.ConclusionsBenchmark data sets and software are crucial tools for the assessment and comparison of competing algorithms. We believe that the miRcomp and miRcompData packages will facilitate the development of new methodology for microRNA expression estimation.

Potential role of cervicovaginal extracellular particles in diagnosis of endometriosis

BackgroundMacaques are an excellent model for many human diseases, including reproductive diseases such as endometriosis. A long-recognized need for early biomarkers of endometriosis has not yet resulted in consensus. While biomarker studies have examined many bodily fluids and targets, cervicovaginal secretions have been relatively under-investigated. Extracellular vesicles (EVs, including exosomes and microvesicles) are found in every biofluid examined, carry cargo including proteins and RNA, and may participate in intercellular signaling. Little is known about EVs in the cervicovaginal compartment, including the effects of reproductive tract disease on quantity and quality of EVs.Case presentationIn September 2014, a 9-year-old rhesus macaque was diagnosed with endometriosis at The Johns Hopkins University School of Medicine. Ultrasound-guided fine needle aspiration of a cyst and subsequent laparotomy confirmed diagnosis. The animal was sent to necropsy following euthanasia for humane reasons. Perimortem vaginal swabs and cervicovaginal lavages were obtained. Using a combination of methods, including ultracentrifugation and NanoSight visualization technology, approximate numbers of EVs from each sample were calculated and compared to populations of EVs from other, reproductively normal macaques. Fewer EVs were recovered from the endometriosis samples as compared with those from reproductively healthy individuals.ConclusionTo our knowledge, this is the first examination of EVs in primate cervicovaginal secretions, including those of a macaque with endometriosis. This case study suggests that additional research is justified to determine whether quantification of EVs—or their molecular cargo—in cervicovaginal lavage and vaginal swabs may provide a novel, relatively non-invasive diagnostic for primate endometrial disease or other reproductive tract diseases.

Synthetic miR-16-5p does not act as a reverse transcriptase co-factor to enhance detection of small RNA

Failure to detect low-abundance microRNAs (miRNAs), for example, in circulating plasma, may occur for a variety of reasons, including presence of enzyme inhibitors. Recently, we received the unusual but intriguing suggestion that miR-16-5p acts as a co-factor of reverse transcriptases, facilitating more efficient reverse transcription of miRNAs and thus enhanced detection of low-abundance miRNAs. We tested this hypothesis by incubating reverse transcriptase with several concentrations of synthetic miR-16-5p and then performing stem-loop RT-qPCR with serial dilutions of miRNA osa-MIR168a. Our results do not support a role for miR-16 as a co-factor of reverse transcriptase.