Journal
MICROCHIMICA ACTA
Volume 184, Issue 11, Pages 4359-4365Publisher
SPRINGER WIEN
DOI: 10.1007/s00604-017-2450-6
Keywords
MicroRNAdetection; Fluorescence assay; Signal amplification; SYBR Green II; DNA polymerase; Breast cancer; Serumsamples
Categories
Funding
- Natural Science Foundation of Shandong province, China [ZR2014BQ029, ZR2017MD023]
- National Natural Science Foundation of China [21375079]
- National Key Research and Development Program of China [2016YFD0800304]
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The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the 10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.
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