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A highly sensitive and selective signal-on strategy for microRNA quantification
- Source :
- Anal Chim Acta
- Publication Year :
- 2019
-
Abstract
- MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all ‘A’ sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation.
- Subjects :
- Analyte
Calibration curve
Cell Culture Techniques
DNA, Single-Stranded
02 engineering and technology
01 natural sciences
Biochemistry
High-performance liquid chromatography
Article
Analytical Chemistry
Limit of Detection
Environmental Chemistry
Animals
Humans
Solid phase extraction
Bovine serum albumin
Locked nucleic acid
Spectroscopy
Polymerase
Chromatography, High Pressure Liquid
Detection limit
Chromatography
biology
Chemistry
010401 analytical chemistry
021001 nanoscience & nanotechnology
0104 chemical sciences
MicroRNAs
Calibration
biology.protein
MCF-7 Cells
Biological Assay
Cattle
0210 nano-technology
Nucleic Acid Amplification Techniques
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Anal Chim Acta
- Accession number :
- edsair.doi.dedup.....0988215832f47940f7ab7ae1c1e04006