Illumina HiSeq 2500 sequencing; microRNA profiling of fetal lamb heart treated with coiled implantation in left atrium against untreated controls
microRNA profiling of fetal lamb heart treated with coiled implantation in left atrium against untreated controls
Under maternal general anesthesia and after fetal echocardiography, fetal lambs were delivered by hysterotomy. The umbilical cord was clamped and the lamb was euthanized with IV sodium pentobarbital (BimedaMTC, Cambridge, Canada). A median sternotomy was used to harvest the heart, then samples were taken from the LV, RV, AAo and main PA. Tissue was snap frozen with liquid N2 and stored at -80°C. At gestational age 76 days, coils were implanted in the left heart under continuous ultrasound guidance (Philips HDI 5000), using a completely percutaneous approach under strict aseptic conditions. A 20G needle (6” Quincke; Beckton-Dickinson) was used to deliver platinum coils (0.018” IDC, Boston Scientific) above the mitral valve until the LV appeared underfilled and the ascending aortic flow decreased or became retrograde. RNA was extracted using the miRNeasy Mini Kit (QIAGEN). Small RNA library preparation was performed following the NEBNext Small RNA Library Prep Set for Illumina protocol. Briefly, 600 ng of total RNA was used to ligate 3' end Illumina-compatible adaptors, followed by hybridization with the reverse transcription primer that prevents formation of adaptor dimers; then 5' end adaptors were ligated to the small RNA, and adapter-ligated small RNA molecules were reverse transcribed and amplified by PCR with an initial denaturation at 94°C for 30 seconds, followed by 15 cycles of 94°C for 15 seconds, 62°C for 30 seconds and 70°C for 15 seconds, and a final extension step for 5 minutes at 70°C. At the amplification step, each sample was amplified with a different barcoded adapter to allow for multiplex sequencing.