LC-MS-Based Direct Quantification of MicroRNAs in Rat Blood

Abstract

MicroRNA (miRNA) has recently garnered significant research attention, owing to its potential as a diagnostic biomarker and therapeutic target. Liquid chromatography-mass spectrometry (LC-MS) offers accurate quantification, multiplexing capacity, and high compatibility with various matrices. These advantages establish it as a preferred technique for detecting miRNA in biological samples. In this study, we presented an LC-MS method for directly quantifying seven miRNAs (rno-miR-150, 146a, 21, 155, 223, 181a, and 125a) associated with immune and inflammatory responses in rat whole blood. To ensure miRNA stability in the samples and efficiently purify target analytes, we compared Trizol- and proteinase K-based extraction methods, and the Trizol extraction proved to be superior in terms of analytical sensitivity and convenience. Chromatographic separation was carried out using an oligonucleotide C18 column with a mobile phase composed of N-butyldimethylamine, 1,1,1,3,3,3-hexafluoro-2-propanol, and methanol. For MS detection, we performed high-resolution full scan analysis using an orbitrap mass analyzer with negative electrospray ionization. The established method was validated by assessing its selectivity, linearity, limit of quantification, accuracy, precision, recovery, matrix effect, carry-over, and stability. The proposed assay was then applied to simultaneously monitor target miRNAs in lipopolysaccharide-treated rats. Although potentially less sensitive than conventional methods, such as qPCR and microarray, this direct-detection-based LC-MS method can accurately and precisely quantify miRNA. Given these promising results, this method could be effectively deployed in various miRNA-related applications.

Figure 1

Effect of the N-butyldimethylamine (DMBA) concentration on detection sensitivity. Peak areas (A) and signal-to-noise (S/N) ratios (B) were normalized relative to the corresponding results obtained from 15 mM DMBA.

Figure 2

Effect of the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) concentration on detection sensitivity. Peak areas (A) and signal-to-noise (S/N) ratios (B) were normalized relative to the corresponding results obtained from 100 mM HFIP.

Figure 3

Liquid chromatography–mass spectrometry (LC–MS) chromatograms of seven miRNAs in (A) a representative blank sample and (B) a blank sample spiked with target miRNAs at the limit of quantification (LOQ, 6.25 nM).