Total rna isolation reagent

Total RNA was extracted using Total RNA Isolation Reagent (Biosharp, Cat #BS259A). Reverse transcription was performed using the FastKing RT Kit (Tiangen, Cat #KR118-02). Quantitative PCR was performed using the Powerup SYBR Master Mix (Applied Biosystems, Cat #A25778). These experiments were conducted according to the corresponding manufacturer’s manuals. Gapdh was used as a normalized control gene. The primer sequences used in real-time quantitative PCR (RT-qPCR) are listed in Supplementary Table 1.

Using the Total RNA Isolation Reagent (BS259A, Biosharp, China), total RNA was extracted from heart tissues or cells. After that, the total RNA underwent reverse transcription and quantification on a Real-Time PCR system (7500, ThermoFisher, USA) using the One-Step SYBR Green qRT-PCR Kit (BL643B, Biosharp, China). Under the adoption of the 2^−ΔΔCT method, B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), major histocompatibility complex-B (B-MHC), GSK3A, CD36, PDK4 (Pyruvate dehydrogenase kinase 4), and Cpt1b (carnitine palmitoyltransferase 1B) expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [12 (link)]. Supplementary Table 3 lists the primer sequences.

Total RNA was isolated using Total RNA Isolation Reagent (Biosharp Life sciences, Beijing, China) according to the manufacturer’s instructions. Reverse transcription was performed using Hifair II 1st Strand cDNA Synthesis SuperMix with a gDNA digester (Yeasen, HB181210, Shanghai, China). Quantitative real-time PCR was performed using SYBR Green Master Mix (Yeasen, HB181203, Shanghai, China) on a Lightcycler Real-Time PCR System (Roche, Beijing, China). The relative expression level of the target genes and the relative fold change were normalized to GAPDH and the control, respectively. The sequences of primers (Tianyihuiyuan, Guangzhou, China) are shown in Table S2.

Total RNA was extracted using Total RNA Isolation Reagent (Biosharp, China) and then reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit (Takara, China). The qPCR was performed using TB Green^® Premix Ex Taq™ II (Takara, China) on the 7,500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). The 2^−ΔCt method was used to calculate gene transcription level, with β-actin mRNA as control. The primer sequences are listed in Table 1.

Total RNA samples were extracted using Total RNA Isolation Reagent (Biosharp, Hefei, China). For protein-coding gene expression quantification, reverse transcriptions were performed using FastKing RT Kit (Tiangen, Beijing, China) and Quantitative PCRs were performed using Powerup SYBR Master Mix (Applied Biosystems, CA, USA). GAPDH served as a normalized control. As to microRNA, cDNA samples were produced sequentially by adding poly(A) tail with E.coli Poly(A) Polymerase (New England Biolabs, MA, USA) and doing reverse transcription with SuperScript™ III Reverse Transcriptase (Invitrogen) and a universal reverse transcription primer (5′-CAGGTCCAGTTTTTTTTTTTTTTTVN-3′; “V” stands for A, C, or G, and “N” stands for A, T, C, or G.) as described previously^{43 (link)–45 (link)}. Quantitative PCRs for microRNA were performed utilizing Powerup SYBR Master Mix (Applied Biosystems). microRNAs′ expression was relative to U6. All quantitative PCR primer sequences are provided in the supplement (Table S1).