Here we present the result of our work on achieving sub-pM limit of quantitation in direct quantitative analysis of multiple miRNAs by capillary electophoresis with laser-induced fluorescence detection. The PDF file contains the description of the results with figures, references, and supporting information. The raw data.ZIP
file is the archive containing (i) Excel files of raw data used to build figures presented in the PDF file and (ii) the red-me.PDF
file explaining how to read and use the Excel files. The figure data.ZIP
file is the archive of all figure files in Origin and Adobe Illustrator formats.
Thousands of putative miRNA-based cancer biomarkers have been reported but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method which is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. Capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard) but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here we explain challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly-negatively charged PNA–miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA–miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers.
ORCID For Submitting Author0000-0003-3270-2130
Declaration of Conflict of InterestThe authors declare no conflict of interest
Version NotesVersion number 1 of August 24, 2020