External Validation of AMI Biomarkers

The GSE59867 dataset, based on the GPL6244 platform, was published by Maciejak et al. [25 (link)] and was used as the independent external validation set, including 111 patients with AMI at admission and 46 patients with stable coronary artery disease. The “limma” package was used to normalize the gene expression profiles [26 (link)].
Whole blood samples were collected from five AMI patients and five normal patients for real-time quantitative polymerase chain reaction (qPCR) to confirm the results. The study was approved by the Ethics Committee of Jiangxi Provincial People’s Hospital, and all patients signed informed consent forms. All patient samples were processed to isolate peripheral blood mononuclear cells immediately after collection and stored at −80°C before RNA extraction. After the samples were pretreated, RNA was extracted using TRIzol reagent (Invitrogen), and qPCR was performed. Total RNA was reverse transcribed into complementary DNA by a qPCR real-time kit (Invitrogen) following the manufacturer’s instructions. Relative gene expression was analyzed by the 2^−ΔΔCT method with normalization to ACTB (internal reference gene). All primers used in this study are shown in Table 1. Data are presented as the mean ± standard deviation. GraphPad Prism 8 software (GraphPad Software, CA) and R software were used for statistical analyses. Analysis of variance or a t test was used for statistical comparisons. P < 0.05 was considered significant.