A piece of LV was placed in ribonuclease-free microcentrifuge tubes containing RLT lysis buffer (QIAGEN) and stainless steel beads (Green Bead Lysis Kit, Next Advance Inc.) and homogenized in a Bullet Blender (BBX24, Next Advance Inc.) at speed 10 for 4 min at 4°C. Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit, including on-column deoxyribonuclease digestion (QIAGEN). cDNA was synthesized from 30 ng of total RNA using the SuperScript IV (Thermo Fisher Scientific). An oligo deoxythymidine dT primer was used to enrich for mature mRNAs, while a random hexamer primer was used to obtain immature mRNAs. Five microliters of template cDNA (diluted 1:6) was used in a PCR with Maxima SYBR Green qPCR master mix (Thermo Fisher Scientific) on a LightCycler 480 (Roche). To determine relative gene expression, a modified ΔΔCt method was used (27). This method takes primer efficiencies (E) into account and normalizes each sample to the geometric mean of multiple reference genes. Reference genes ribosomal protein L32 (Rpl32) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), shown to be stable in failing human hearts, were selected from the literature (28, 29). Primer efficiencies were determined from standard curves generated using cDNA dilutions. Primers included Lmod2 spanning intron 1 [5′-GGACATTCAGCAGAGAGGCACT-3′ (forward) and 5′-GATAAGCTCTTCTTCACTTTCCTC-3′ (reverse); 128-bp product; E = 1.834], Lmod2 located within intron 1 [5′-TGCAGATAGAGCCAGAGGGT-3′ (forward) and 5′-GGGGCCTGTCTCAAAATCCA-3′ (reverse); 95-bp product; E = 1.873], Rpl32 [5′-CACCAGTCAGACCGATATGTCAAAA-3′ (forward) and 5′-TGTTGTCAATGCCTCTGGGTTT-3′ (reverse); 64-bp product; E = 1.912], and YWHAZ [5′-ACCGTTACTTGGCTGAGGTTGC-3′ (forward) and 5′-CCCAGTCTGATAGGATGTGTTGG-3′ (reverse); 130-bp product; E = 2.064].

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