This may indicate the existence of single-cell miRNA expression heterogeneity but could be due in part to the limit of detection (LOD) our method can provide as of now

This may indicate the existence of single-cell miRNA expression heterogeneity but could be due in part to the limit of detection (LOD) our method can provide as of now. of microtroughs. It offers high throughput and high multiplexing capability for evaluating microRNA heterogeneity in Rabbit polyclonal to ANKRD49 single cells, representing a new approach toward microRNA-based diagnosis and RCGD423 monitoring of complex RCGD423 human diseases. Keywords: single-cell analysis, microRNA, DNA barcoding, microtrough arrays 1. Introduction MicroRNAs (miRNAs), an important class of small RNAs, are involved in nearly all biological processes including cell proliferation, differentiation, and metabolism [1,2,3,4,5]. They regulate messenger RNA (mRNA) expression level or function via mRNA translation repression or RNA degradation [1]. Dysregulated apoptosis or abnormal proliferation of cells, often implicated in human cancers, can be induced by miRNAs [6,7,8]. Deletion of tumor-suppressive miRNAs (TS-miRs) or over expression of oncogenic miRNAs (onco-miRs) have been identified in several types of cancer [9,10,11]. However, due to the complex regulatory mechanism of miRNAs in modulating target mRNAs, how to understand the exact role of each miRNA in every cell and how to translate such findings to a miRNA biomarker assay for clinical diagnostics is a challenging task. In particular, due to non-genetic cell-cell heterogeneity in development or tumors [3,5,12], it is required to conduct multiplexed miRNA measurements at the level of single cells. To date, despite the recent progress in single-cell small RNA sequencing [13,14], how to measure a panel of miRNAs in a highly-multiplexed manner from hundreds to thousands of single cells such that intratumor miRNA heterogeneity can be quantitatively examined with high statistical power, is still difficult. Moreover, how to perform such assays in the clinical settings via a rapid on-site test is a challenge that needs to be overcome to bring single cell miRNA profiles to cancer diagnosis and classification. A majority of existing microRNA detection technologies such as RT-PCR or microarray-based miRNA assays are not suitable for single cell analysis [15,16]. The PreAmp RCGD423 workflow (ABI/Thermo Fisher, Waltham, MA, USA) adds an intermediate step between RT and real-time PCR to pre-amplify cDNA prior to qPCR detection RCGD423 [17]. However, it is mainly for single-plex real-time PCR, and each PCR tube contains one cell, which is a low throughput and labor intense process. AmpliGrid microwell array-based solitary cell capture chip allows for medium throughput miRNA assay on 48 solitary cells per chip [18]. However, this technology is definitely expensive and offers very limited multiplexing ability. To execute the conventional RNA processing workflow at the level of solitary cells requires miniaturized fluid handling systems to conduct multi-step RNA extraction, chemical changes, purification, and amplification, for example, inside a Fluidigm C1? (South San Francisco, CA, USA) Single-Cell Auto Prep System [19]. It is probably one of the most powerful systems for complex biochemical workflow in the nanoliter level and together with the BioMark HD System allows for control of ~96 solitary cells for RCGD423 any panel of genes [20]. However, this device is definitely costly, and the workflow is definitely time consuming. A microfluidic chip was reported for analyzing >1000 solitary cells for on-chip qRT-PCR detection of 1 1 or 2 2 miRNA biomarkers simultaneously but is still yet to demonstrate highly multiplexed detection. As of today, these systems either lack the ability of a highly multiplexed assay or only gives low to medium throughput. Moreover, most of these methods are time-consuming, expensive, or clinically impractical. Here, we statement on a sub-nanoliter microtrough array chip for multiplexed and high-throughput miRNA detection in the single-cell level. The microtroughs are used to encapsulate solitary cancer cells that were fixed, permeabilized, and pre-incubated with a set of distinctively designed miRNA detection probes, each of which consists of a DNA oligomer capture strand complementary to the miRNA of interest and the other half is definitely a unique reporter strand that can be cleaved inside the microtrough upon UV exposure. The cleaved reporter strands are then captured by an array of DNA barcodes pattern on the bottom of each microtrough such that the detection of reporter strands with known sequences is definitely a surrogate for detecting miRNAs in solitary cells via.