Hybond n nitrocellulose membrane

For the analysis of global hydroxymethylation levels, genomic DNA was isolated from mouse brains, ESCs, and small intestinal epithelia using the Allprep kit (Qiagen). dsDNAs were denatured for 10 min at 95°C and spotted onto Hybond-N⁺ nitrocellulose membranes (GE Healthcare). The membrane was blocked with CAS-Block (Thermo Fisher Scientific) followed by overnight incubation at 4°C with an anti-5hmC antibody (Active Motif). Membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (GE Healthcare) for 30 min at room temperature and developed using the ECL⁺ prime blotting detection system (GE Healthcare).

RNA was isolated from total hearts or heart mitochondria, and northern blotting and qRT-PCR were performed as described previously (18 (link), 41 (link)). RNA was isolated using the miRNeasy Mini Kit (Qiagen) incorporating an on-column RNase-free deoxyribonuclease (DNase) digestion to remove all DNA. RNA (3 μg) was resolved on 1.2% agarose formaldehyde gels, then transferred to 0.45-μm Hybond-N⁺ nitrocellulose membrane (GE Lifesciences), and hybridized with biotinylated oligonucleotide probes specific to mouse mt-mRNAs, rRNAs, and tRNAs (42 (link)). Hybridizations were carried out overnight at 50°C in 5× SSC, 20 mM Na₂HPO₄, 7% SDS, and heparin (100 μg ml⁻¹), followed by washing. The signal was detected using streptavidin-linked infrared-labeled antibody [diluted 1:2000 in 3× SSC, 5% SDS, and 25 mM Na₂HPO₄ (pH 7.5)] using the Odyssey Infrared Imaging System (Li-Cor). Complementary DNA (cDNA) was prepared using the QuantiTect Reverse Transcription Kit (Qiagen) and used as a template in the subsequent PCR that was performed using a Corbett Rotor-Gene 6000 instrument with SensiMix SYBR mix (Bioline) and normalized to 18S rRNA.

The tissues were snap-frozen and stored at –80°C before RNA isolation, while pancreatic islets were isolated fresh using collagenase digestion as described below. RNA was isolated from tissues and pancreatic islets of Langerhans using the miRNeasy Mini kit (Qiagen) using an on-column RNase-free DNase digestion, and the isolated RNA (4 μg) was resolved on 1.2% agarose formaldehyde gels. Northern blotting was carried out as described previously (13 (link)) by transferring the RNA to 0.45 μm of Hybond-N⁺ nitrocellulose membrane (GE Lifesciences) and hybridizing using biotinylated oligonucleotide probes as described before (13 (link)). Streptavidin-linked infrared-labeled antibody [diluted 1:10,000 in 3× SSC, 5% SDS, and 25 mM Na₂HPO₄ (pH 7.5)] was used to detect the signal and visualize the signal using the Odyssey Infrared Imaging System (LI-COR Biosciences). The QuantiTect Reverse Transcription Kit (Qiagen) was used to prepare complementary DNA (cDNA) that was used as a template for PCR. PCRs were performed using a Corbett Rotorgene 6000 using SensiMix SYBR mix (Bioline) and normalized to 18S rRNA.