Lysing matrix d 1.4 mm ceramic beads

For RNA isolation, hearts and pancreata stored at  − 80 °C were used. Approximately 20–30 mg of tissue were transferred to the RLT buffer and homogenized with a FastPrep96 system as recommended (Lysing Matrix D 1.4-mm ceramic beads; MP Biomedicals, Irvine, CA). RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany), and samples were treated with deoxyribonuclease (DNase) I and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). In a single-step reaction, RNA was reverse-transcribed and PCR was performed using the iTaq Universal one-step RT-qPCR kit (BioRad, Hercules, CA). The real-time quantitative PCR analysis included amplifications for CVB3 VP1 (target gene) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, house-keeping gene) using TaqMan Gene Expression Assays (Applied Biosystems) and the CFX96 Touch Real-time PCR detection system (BioRad). Expression of CVB3 VP1 was normalized to GAPDH using the 2^{−(∆∆Ct)} method.

Pancreata were collected during termination and stored at −80 °C until RNA isolation. Approximately 30 mg of pancreatic tissue was homogenized with the FastPrep-96™ instrument as recommended (Lysing Matrix D 1.4 mm ceramic beads; MP Biomedicals, Irvine, CA, USA). Total RNA was isolated using the PureLink™ RNA Mini Kit (Invitrogen, Waltham, MA, USA), and samples were treated with deoxyribonuclease I and quantified using the NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific). In a single-step reaction, RNA was reverse-transcribed, and PCR was performed using the PrimeTime™ One-Step RT-qPCR Master Mix (Integrated DNA Technologies, Coralville, IA, USA). Amplifications were performed using the CVB3 VP1 [target gene] (forward, 5′-TTGCATATGGCCCAGTGGAAG-3′; reverse, 5′-TGTGGATCCTTATTGCCTAGTAGTGGTAACTC-3′) and glyceraldehyde-3-phosphate dehydrogenase [GAPDH, housekeeping gene] (forward, 5′-CGGCAAATTCAACGGCACAGTCAA-3′; reverse, 5′- CTTTCCAGAGGGGCCATCCACAG-3′) TaqMan^® Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) [40 (link)] and the CFX96 Touch Real-time PCR Detection System thermocycler (BioRad, Hercules, CA, USA). Expression of CVB3 VP1 was normalized to GAPDH using the 2^{−(∆∆Ct)} method as reported previously [33 (link)].

Approximately 20–30 mg of heart, thymus, and spleen tissue harvested from various strains of mice were snap-frozen in liquid nitrogen and stored at −80 °C. For RNA isolation, tissues were transferred to lysis buffer containing β-mercaptoethanol and homogenized using the FastPrep-24™ system, according to the manufacturer’s recommendations (Lysing Matrix D 1.4-mm ceramic beads; MP Biomedicals, Irvine, CA, USA). RNA was extracted using the RNA Mini Kit (PureLink™, Thermo Fisher Scientific, Waltham, MA, USA) and eluted in RNase-free water. The isolated RNA samples were treated with deoxyribonuclease (DNase) I and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). In a single-step reaction, 50 ng of RNA was reverse-transcribed, and qPCR was performed using the iTaq™ Universal SYBR^® Green One-Step Kit. Real-time qPCR analysis was performed using sequence-specific primers for mouse Myhc-α (5′-GCTACACTCTTCTCTACC-3′ and 5′-CATAGAGAATGCGGTTGG-3′) and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′CTCCCACTCTTCCACCTTCG-3′ and 5′GCCTCTCTTGCTCAGTGTCC-3′). The CFX96 Touch Real-time PCR detection system (BioRad, Hercules, CA, USA) was used for thermocycling, and the relative normalized gene expression of mouse cardiac myosin was analyzed using the 2^−(ΔΔCt) method [21 (link)] in BioRad CFX Maestro 1.0 software.