Various methods are used to analyze contents in scientific setups quantitatively. This article summarizes a research article for the analysis of micro ribonucleic acid miR-451 from plasma using liquid chromatography-mass spectrometry (Basiri et al. 2019). The paper explores the method used, the matrix, and the nature of the analyte, the specific conclusions and an appraisal of the quality of analysis as well as the sampling statistics and technique. Establishing clear linear quantitative patterns for plasma concentration of mRNA could be a critical diagnostic tool that can be used to asses level of recovery or gravity of a given disease state. Cancer therapy efficacy can be evaluated using Micro RNAs as the markers. There has not been an ideal quantitative tool for analysis. The article is the first mass spectrometry-based quantitation of mir-451. MiR-451 which is a highly conserved micro RNA in most vertebrates is the specific species being analyzed. In the body, MiR-451 is used for the regulation of erythroid maturation. It is thus used as an indicator of erythroid related conditions such as the management of polycythaemias. The quantities of miR-451 are highly irregular in a couple of human cancers such as lung cancer, leukemia, and hepatocellular carcinoma. A stock solution containing 1mg/ml of miR-451 was prepared in nuclease-free water and stored at -80°C. Proteinase K buffer and lipase were used in the digestion buffer for the nuclease. Serial dilution was prepared. All surfaces coming into contact with the sample were cleaned with RNaseZAP to ensure no exterior RNA come into contact with the analyte sample. DNA lobind tubes were used for analysis to ensure no losses of the RNA to the tube walls.
The analysis was done using a liquid chromatography-mass spectrometer whose specifications are indicated in the article. Liquid Chromatography is an analytical technique that helps separate the various contents within a mixture matrix. The different solubility of the different components of the array makes this possible. After exiting the liquid chromatography column, the separated analytes go into the mass spectrometer. The mass spectrometer helps determine the quantities of the analytes; this happens by separating the ions of the analytes through various ionization methods, such as chemical ionization and electron impact ionization. The analytes are then separated as per their molecular masses using magnetic and electric fields. The masses obtained are then compared with libraries, and besides quantitation, mass spectrometers can be used for identification. The limit of quantitation which is the lowest measurable quantity was determined to be 0.5nh/ml. The precision of the method was determined by performing an analysis of four different concentrations. The accuracy was determined by performing the analysis along with a concentration curve. All the two values fall within the allowed FDA ranges with the highest standard deviation for precision at 19.5% and least at 7.6%. Accuracy was at lowest 12.9% and the most top 20%. The measured quantities for the four samples in ng/ml were 0.6, 1.1, 22.7 and 174.2. The above article represented a successful analysis of mRNA using a new mobile phase made of 15 mM DBA and 25 mM HFMIP in LC-MS. The method is of top-notch quality noting the values obtained in both the precision and accuracy tests. The use of DNAseZap and lobind tubes also greatly minimizes error. The sample size is large enough to allow sufficient sample preparation. Adherence to standard operating procedures especially with the processing procedures for nucleic acid materials and temperatures was well adhered to in the study. The study not only demonstrated statistical significance, but it also presented justifiable results that were deduced amid negligible limitations.
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References
Basiri, B., Sutton, J.M., Hooshfar, S., Byrnes, C.C., Murph, M.M. and Bartlett, M.G., 2019. Direct identification of microribonucleic acid miR-451 from plasma using liquid chromatography-mass spectrometry. Journal of Chromatography A , 1584 , pp.97-105.
