Quantitative Reverse Transcriptase Polymerase Chain Reaction

Total RNA was exacted using TRIzol reagent (Qiagen, Valencia, CA, USA) and used in reverse-transcription and PCR amplification reactions. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR production were assessed to ensure that equal amounts of RNA were input to the RT-PCR reaction. Real-time quantitative RT-PCR was performed using the CFX96 Real-Time PCR System with specific primers and software (Bio-Rad, Hercules, CA, USA). For miRNA studies, we quantified the level of miR-223 in cells and tissues with real-time quantitative RT-PCR, and small nuclear RNA U6 was used as an endogenous control. We also tested the level of miR-223 in the serum of heart transplant patients and recipient mice, and cel-miR-39 was used as an endogenous control. All primers, cel-miR-39, and miRNA mimics and inhibitors were purchased from RiboBio (Guangzhou, China). The primer sequences used for RT-PCR are shown in Table 1.

Primer sequences used for RT-PCR.