MicroRNA Detection: Challenges for the Analytical Chemist

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

“…4,5 Given these constraints, a clinically-viable microRNA detection assay will require picomolar or better sensitivity (e.g., assuming 10 000 cells, or approximately 100 ng of total RNA, as the starting sample), greater than 3 orders of magnitude dynamic range, and single-nucleotide specificity, in addition, the quantification and delivery of an answer in a relatively short amount of time. Currently, sensitive microRNA detection is typically performed with qPCR, which boasts nearsingle-molecule sensitivity, high selectivity, and 10 7 -fold dynamic range.…”

“…4,5,8−10 In contrast, traditional northern blotting is highly-quantitative yet takes days to complete and requires large amounts of sample (∼10 μg of total RNA or approximately 10 6 cells). 4 Microarrays are capable of a large degree of multiplexing, absolute quantification, and high sensitivity (in the 1 fg range) but require incubation for hours or days to achieve these limits.…”

“…4 We here present an assay that integrates ITP-aided hybridization with a functionalized hydrogel matrix to overcome these drawbacks. ITP is an electrokinetic focusing technique that utilizes a heterogeneous buffer system, composed of a leading (LE) and trailing electrolyte (TE), to…”

“…1 Dysregulation of microRNA expression has been linked to multiple forms of cancer and is considered an important diagnostic and prognostic signature. 2−5 Absolute quantification of microRNA species presents four main challenges: (1) microRNAs are low in abundance, together making up only about 0.1% (w/w) of a cell's total RNA content, 6 (2) concentrations of a single microRNA species can vary by over 3 orders of magnitude, 7 (3) microRNA species can differ by as little as a single nucleotide, 4,5 and (4) so-called "mature" microRNA species must be distinguished from "precursors" sharing the full mature sequence. 4,5 Given these constraints, a clinically-viable microRNA detection assay will require picomolar or better sensitivity (e.g., assuming 10 000 cells, or approximately 100 ng of total RNA, as the starting sample), greater than 3 orders of magnitude dynamic range, and single-nucleotide specificity, in addition, the quantification and delivery of an answer in a relatively short amount of time.…”

“…4,5,8−10 In contrast, traditional northern blotting is highly-quantitative yet takes days to complete and requires large amounts of sample (∼10 μg of total RNA or approximately 10 6 cells). 4 Microarrays are capable of a large degree of multiplexing, absolute quantification, and high sensitivity (in the 1 fg range) but require incubation for hours or days to achieve these limits. 11−13 Sequencing is emerging as a unique platform for small RNA discovery but is ill-suited to diagnostics because it is sample-hungry, requires expensive equipment and reagents, and can take up to 2 weeks to complete.…”