Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Wong, Wilson; Farr, Ryan J.; Joglekar, Mugdha V.; Januszewski, Andrzej S.; Hardikar, Anandwardhan A.

doi:10.3791/52586

Cited by 30 publications

(23 citation statements)

References 31 publications

(32 reference statements)

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“…We found a comparable level of reproducibility on this platform when using cards from the same lot ( Supplementary Fig. 1 ), but have observed considerable lot-to-lot variation 29 .…”

Section: Discussionmentioning

confidence: 54%

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

Farr

Januszewski

Joglekar

et al. 2015

Sci Rep

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MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultra-high content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest.

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“…We found a comparable level of reproducibility on this platform when using cards from the same lot ( Supplementary Fig. 1 ), but have observed considerable lot-to-lot variation 29 .…”

Section: Discussionmentioning

confidence: 54%

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

Farr

Januszewski

Joglekar

et al. 2015

Sci Rep

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“…RNA isolation methods were evaluated for maximum RNA yield and amplifiable miRNA levels. Isolation method efficiency was measured through RT‐qPCR analysis, as previous work has shown that UV spectrophotometry and other methods cannot precisely predict miRNA concentrations in the low concentrations observed in biological fluids . Consistent volumes of biological secretions from the same donor and donation were used, and elution volumes were normalized to 50 μL to allow for ease of comparison.…”

Section: Resultsmentioning

confidence: 99%

High‐throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids

et al. 2016

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microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina Hi-Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body fluid specific (miRs-200b, 1246, 320c, 10b-5p, 26b, and 891a) and potential normalization miRNAs (let-7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid.

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“…RNA extraction, cDNA synthesis, and preamplification were performed as previously described (10). Human Pool A v2.1 and B v3.0 Megaplex stem-loop RT Primers (Applied Biosystems) were used to produce cDNA.…”

Section: Mirna Profilingmentioning

confidence: 99%

Coxsackievirus B5 Infection Induces Dysregulation of microRNAs Predicted to Target Known Type 1 Diabetes Risk Genes in Human Pancreatic Islets

Kim

Alshabee-Akil

et al. 2015

Diabetes

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Extensive research has identified enterovirus (EV) infections as key environmental triggers of type 1 diabetes. However, the underlying molecular mechanisms via which EVs contribute to the pathogenesis of type 1 diabetes remain unclear. Given that EVs dysregulate host microRNAs (miRNAs), which function as key regulators of β-cell biology, we investigated the impact of coxsackievirus B5 (CVB5) infection on the cellular expression of miRNAs within human islets. Using high-throughput quantitative PCR nanofluidics arrays, the expression of 754 miRNAs was examined in CVB5-infected human pancreatic islets. In total, 33 miRNAs were significantly dysregulated (≥ threefold difference) in the infected compared with control islets (P < 0.05). Subsequently, these differentially expressed miRNAs were predicted to target mRNAs of 57 known type 1 diabetes risk genes that collectively mediate various biological processes, including the regulation of cell proliferation, cytokine production, the innate immune response, and apoptosis. In conclusion, we report the first global miRNA expression profiling of CVB5-infected human pancreatic islets. We propose that EVs disrupt the miRNA-directed suppression of proinflammatory factors within β-cells, thereby resulting in an exacerbated antiviral immune response that promotes β-cell destruction and eventual type 1 diabetes.

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Cited by 30 publications

References 31 publications

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

High‐throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids

Coxsackievirus B5 Infection Induces Dysregulation of microRNAs Predicted to Target Known Type 1 Diabetes Risk Genes in Human Pancreatic Islets

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