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

Abstract

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.

Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas

“…The differences between TLDA and RT-qPCR protocols (pooled reverse transcription in TLDA vs. individual RT and qPCR; preamplification used in TLDA measurement vs. without preamplification; differences in the reaction volume 1 μl in TLDA vs. 15 μl in individual RT-qPCR) can influence the efficiency of both the RT and qPCR leading to this discrepancy that was found between the two approaches. Also, high variance of the replicates at TLDA system (median: 8.3%; min-max: 0.3-19.1%) has been also described in literature [25].…”

Biomarkers in Diabetic Retinopathy

“…For example, we recently published a comparison of five different platforms to detect and quantify a microRNA signature for diabetic retinopathy. The different platforms were associated with different sensitivities, sample volumes required, and cost per sample [85]. Maintenance should be kept up to date as poorly maintained or faulty instruments can generate erroneous results.…”

“…Our team is currently taking a discovery rather than a candidate approach to microRNAs for latestage diabetic retinopathy, and recently published a paper highlighting the impact of the instrumentation used for detection of these molecular biomarkers [85]. This new research area holds considerable clinical potential for diagnosis, prognosis, and identifying of potential drug-targets, and even for microRNAs being therapeutic agents themselves.…”

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

“…The result indicated that the synthetic mimics can be successfully discriminated by their respective primer pairs, either the forward primer hsa-miR18a-5p-F/reverse primer hsa-miR-18a-5p-R set or the forward primer hsa-miR-18b-5p-F/reverse primer hsa-miR-18b-5p-R set ( Figure 3a). Despite the range of the Cq value, (this type of range is often observed in nano-volume qPCR platforms (Farr et al 2015), the experiment served the purpose of illustrating the specificity of the primer pairs. In another illustration, the primers designed by specific-FR-system FRM for hsa-miR-16 family members, hsa-miR-16-5p and hsa-miR-195-5p, can distinguish synthetic hsa-miR-16 family oligonucleotide template mimics (Figure 3b).…”