Plasma miRNA Profiling of Cardiovascular Health

Sodium citrate plasma samples were stored at −80 °C until assayed. The FirePlex^® cardiology panel (ABCAM, Cambridge, MA, USA) was employed to measure the plasma levels of 65 miRNAs which have been associated with cardiovascular health. The method has been described previously [24 (link)]. Briefly, 40 μL plasma sample was mixed with Digest Buffer and Protease Mix to a final volume of 80 μL and then incubated at 60 °C for 45 min. A reaction mixture (25 μL of the prepared sample + 35 μL FirePlex Particles + 25 μL of hybridization buffer) was incubated at 37 °C for 60 min in a 96-well plate. After rinsing to remove unbound RNA, 75 μL of labeling buffer was added to each well and incubated for 60 min at room temperature. Adapted-modified miRNAs were eluted using 95 °C water and collected for PCR amplification using a PCR master mix. PCR product was then hybridized with hybridization buffer at 37 °C for 30 min. Particles were then rinsed and scanned on an EMD Millipore Guava 8HT flow cytometer. Raw output was background subtracted, normalized using the geometric mean of three normalizer miRNAs (miR-15b-5p, miR-17-5p, and miR-93-5p).