Analysis of the Distribution Profiles of Circulating MicroRNAs by Asymmetrical Flow Field Flow Fractionation.

MicroRNAs (miRNAs) are stably present in circulatory systems. They are bound to various carriers like proteins, lipoprotein particles, and exosomes. Investigating the process of miRNA distribution among these carriers will help improve our understanding of their functions in the extracellular environment and their potential relationship with diseases. Here, we describe how to obtain the distribution profiles of circulating miRNAs by separation of different miRNA carriers in human serum with asymmetrical flow field flow fractionation (AF4), and detection of the miRNAs in the eluted fractions that enrich particular types of carriers with RT-qPCR.

Distribution profiling of circulating microRNAs in serum.

Circulating microRNAs (miRNAs) are potential biomarkers useful in cancer diagnosis. They have been found to be bound to various carriers like proteins, lipoprotein particles, and exosomes. It is likely that only miRNAs in particular carriers, but not the overall quantity, are directly related to cancer development. Herein, we developed a method for rapid separation of different miRNA carriers in serum using asymmetrical flow field flow fractionation (AF4). Sera from two healthy individuals (control) or from two cancer patients (case) were fractionated. Six fractions enriching different types of miRNA carriers, such as the lipoprotein particles and exosomes, were collected. The quantities of eight selected miRNAs in each fraction were obtained by RT-qPCR to yield their distribution profiles among the carriers. Larger changes in miRNA quantity between the control and the case were detected in the fractionated results compared to the sum values. Statistical analysis on the distribution profiles also proved that, the quantities of 4 miRNAs within particular fractions showed significant difference between the controls and the cases. On the contrary, if the overall quantity of the miRNA was subject to the same statistical analysis, only 2 miRNAs exhibited significant difference. Moreover, principle component analysis revealed good separation between the controls and the cases with the fractionated miRNA amounts. All in all, we have demonstrated that, our method enables comprehensive screening of the distribution of circulating miRNAs in the carriers. The obtained distribution profile enlarges the miRNA expression difference between healthy individuals and cancer patients, facilitating the discovery of specific miRNA biomarkers for cancer diagnosis.

Tagging the rolling circle products with nanocrystal clusters for cascade signal increase in the detection of miRNA.

A new method to label and detect the long ssDNA products from rolling circle (RC) amplification was reported. ZnS nanocrystal clusters (NCCs) were used to tag the RC products. The NCCs release millions of cations to trigger millions of metal-responsive dyes for cascade signal amplification. Selective and sensitive quantification of miRNA was demonstrated; and the NCCs delivered much lower detection limits compared to using peroxidase or quantum dots as the labels.

Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.

The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 µL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.