Microfluidic-based enzymatic on-chip labeling of miRNAs

Abstract

Small noncoding RNAs (sncRNAs) have moved from oddity to recognized important players in gene regulation. Next generation sequencing approaches discover more and more such molecules from a variety of different groups, but flexible tools translating this sequence information into affordable high-throughput assays are missing. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs on highly flexible microfluidic microarrays which combines several beneficial parameters: it can effortless incorporate any new sequence information; it is sensitive enough to work with as little as 20ng of total RNA and has a high level of specificity owing to a combination of a conventional hybridization assay and an enzymatic elongation step. Importantly, no labeling step is needed before hybridization and - because of its high sensitivity - no amplification is required. Both aspects ensure that no bias is introduced by such processes. Although the assay is exemplified with miRNAs, the flexibility of the technology platform allows the analysis of any type of sncRNA, such as piRNAs.

NcRNA-microchip analysis

“…The hybridization temperature, starting at 70°C, was reduced in 2-3°C increments per hour to a final hybridization temperature of 39°C over a period of 16 h. Microchip hybridization was performed on the Febit Geniom technology system. 20,21 NcRNA genes that exhibited signal intensities three times above hybridization background, in at least three of five biological replica, were included in this analysis. Thereby, differential expression of ncRNA genes, indicated as fold change (FC), was calculated by the ratio of hybridization signals of EBV-immortalized versus non-infected ncRNAs.…”

“…16 In total, 3,956 DNA oligonucleotide probes were designed using the Febit Geniom Client software (Table 1). 20,21 Preparation of size-fragmented RNAs is described below (see Results). Labeling was performed using the mirVana TM miRNA labeling kit from Ambion (Austin, TX).…”

“…Microchip data analysis was performed as previously described. 21 Novel ncRNA candidates, which exhibited a signal intensity above 1,000, i.e., three times over the background signal, in at least three of five biological replica, were selected and considered to be expressed. The fold change (FC) of probes of EBV-infected versus non-infected RNA preparations was calculated as a logarithmic value (log2).…”

3D printed microfluidic devices with integrated valves

“…1 is a critical technology for an extremely broad range of biomedical applications including tissue engineering, 2,3 drug discovery, 4 point-of-care diagnostics and pathogen detection in both developed and developing countries, [5][6][7][8] and cancer screening using approaches such as cell identification, 9 and protein, [10][11][12][13] DNA, 14 and micro-RNA 15,16 biomarkers. Microfluidic device prototyping for proof-of-principle demonstration typically utilizes hot embossed or injection molded plastics 1,17 or polydimethylsiloxane (PDMS).…”

Refining Diagnostic MicroRNA Signatures by Whole-miRNome Kinetic Analysis in Acute Myocardial Infarction

“…However, these were single point measurements and blood samples, obtained at different times after the onset of symptoms. Therefore, we embarked on the current proof-of-concept study to investigate the kinetic changes of the whole blood miRNome in the early stage of suspected myocardial infarction at 5 predefined time points using microfluidic primer extension arrays and to replicate the results by using an independent methodology in an independent cohort of early-stage AMI patients (22 ). We hypothesized that miRNA patterns may not only indicate myocardial cell necrosis, as reflected by cTnT, but also provide independent, early diagnostic information.…”