Abstract
BackgroundMicroRNAs (miRNAs) can be used for early diagnosis of myocardial infarction. However, due to a lack of standardized operating procedures, their value for clinical application is low.MethodsDetection of plasma miRNAs was optimized by analyzing factors influencing miRNA variance and myocardial infarction risk scores during analysis (extraction, reverse transcription, and real‐time PCR) and pre‐analysis (dietary status, anticoagulants, storage conditions, and hemolysis).ResultsRegarding variable factors during analysis, the centrifugal column method was superior to Trizol LS reagent when extracting miRNA from plasma. Recovery rate was highest with plasma volumes of 200 and 300 µL. During analysis, the main source of miRNA detection inaccuracy was derived from RNA extraction (mainly organic extraction), and not reverse transcription or PCR. MiRNA variance could be reduced by use of an internal reference. During analysis, 95% of risk score variation fluctuated within a range of 6.267. The variable factors pre‐analysis mainly involved dietary status, anticoagulant selection, and storage conditions. Hemolysis positively correlated with miRNA levels, but there was no significant change in risk score after internal reference calibration.ConclusionPreliminary standardization for miRNA detection provides a reference for clinical blood testing of miRNAs.
Highlights
Many studies have shown that microRNAs can be used for early diagnosis and prognosis of myocardial infarction, but because the detection technology is still developing, it has been difficult to promote use of the approach in the clinic.[1]
This study examined nonspecific fluctuations of current miRNA de‐ tection systems using variables of miRNA detection performance and reproducibility
The RNA extraction step was the main source of inaccuracy, not reverse transcription or PCR.[7]
Summary
Many studies have shown that microRNAs (miRNAs) can be used for early diagnosis and prognosis of myocardial infarction, but because the detection technology is still developing, it has been difficult to promote use of the approach in the clinic.[1] Many vari‐ ables cause variability in miRNA detection, and lack of system‐ atic analysis of these variables exacerbates the problem. MiRNAs are endogenous noncoding small‐mole‐ cule single‐stranded RNAs that regulate gene expression at the post‐transcriptional level.[2] MiRNAs regulate growth and partic‐ ipate in many physiological and pathological processes, includ‐ ing cardiovascular disease.[3] As miR‐126 and miR‐92a are widely present in endothelial cells (EC) and endothelial progenitor cells, their levels are significantly reduced in the plasma of patients with myocardial infarction.[4] we detected varia‐ tion in miR‐126 and miR‐92a levels to identify factors affecting. | 2 of 7 quantification of miRNA. We provide information on testing and improvements to optimize and standardize miRNA detection for clinical application
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