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). Accuracy and precision of miRNA quantitation were determined for miRNA samples in a crude cell lysate, RNA extract from the lysate, and a pure buffer. The results showed that the measurements were matrix-independent with inaccuracies of below 13% from true values and relative standard deviations of below 11% from the mean values in a miRNA concentration range of 2 orders of magnitude. We compared DQAMmiR-derived results with those obtained by a benchmark miRNA-quantitation method-quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR-based measurements revealed multifold inaccuracies and relative standard deviations of up to 70% in crude cell lysate. Robustness of DQAMmiR to changes in sample matrix makes it a perfect candidate for validation and clinical use of miRNA-based disease biomarkers.
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Liang
0000-0003-4507-8525
L
Hu
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Alexander S
AS
Stasheuski
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
David W
DW
Wegman
Sunnybrook Research Institute and Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
Nan
N
Wu
Sunnybrook Research Institute and Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
Burton B
BB
Yang
Department of Biology and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Heyam
H
Hayder
Department of Biology and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Chun
C
Peng
Sunnybrook-Odette Cancer Centre and Department of Radiation Oncology, Sunnybrook Health Sciences Centre , 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.
Stanley K
SK
Liu
Keenan Research Centre, St. Michael's Hospital , 30 Bond Street, Toronto, Ontario M5B 1W8, Canada.
George M
GM
Yousef
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Sergey N
0000-0003-3270-2130
SN
Krylov
D015342:DNA Probes / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D060888:Real-Time Polymerase Chain Reaction
D015342:DNA Probes; D035683:MicroRNAs