Accurate MicroRNA Analysis in Crude Cell Lysate by Capillary Electrophoresis-Based Hybridization Assay in Comparison with Quantitative Reverse Transcription-Polymerase Chain Reaction.

Accurate quantitation of microRNA (miRNA) in tissue samples is required for validation and clinical use of miRNA-based disease biomarkers. Since sample processing, such as RNA extraction, introduces undesirable biases, it is advantageous to measure miRNA in a crude cell lysate. Here, we report on accurate miRNA quantitation in crude cell lysate by a CE-based hybridization assay termed direct quantitative analysis of multiple miRNAs (DQAMmiR).

Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR).

Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact.

Highly-sensitive amplification-free analysis of multiple miRNAs by capillary electrophoresis.

Sets of deregulated microRNAs (miRNAs), termed miRNA signatures, are promising biomarkers for cancer. Validation of miRNA signatures requires a technique that is accurate, sensitive, capable of detecting multiple miRNAs, fast, robust, and not cost-prohibitive. Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a capillary electrophoresis (CE)-based hybridization assay that was suggested as a methodological platform for validation and clinical use of miRNA signatures.

Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust.

Universal drag tag for direct quantitative analysis of multiple microRNAs.

Using microRNA (miRNA) as molecular markers of diseases requires a method for accurate measurement of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed based on a classical hybridization approach. In DQAMmiR, miRNAs are hybridized with excess fluorescently labeled complementary DNA probes.

Ultrasensitive on-column laser-induced fluorescence in capillary electrophoresis using multiparameter confocal detection.

We report a simple method to efficiently couple on-column, standard Capillary Electrophoresis with Confocal MultiParameter Fluorescence detection (CE-CMPF) using only commercially available components. A molecular collection of 13% and a concentration limit of detection of 1.5 pM fluorescein are achieved in our instrument by gating the arrival time of individual photons in order to reduce the scattering contribution.

Detection of a thousand copies of miRNA without enrichment or modification.

We report direct quantitative analysis of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to determine levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7).

Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase.

2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules.

Making DNA hybridization assays in capillary electrophoresis quantitative.

A single-stranded DNA-binding protein (SSB) has been recently proven to facilitate highly efficient separation of the excess hybridization probe from the probe-target hybrid in gel-free capillary electrophoresis. SSB added to the electrophoresis run buffer binds the single-stranded DNA probe but does not bind the double stranded DNA-DNA or DNA-RNA hybrid. As a result, SSB changes the electrophoretic mobility of the probe but does not affect the mobility of the hybrid.