Epigenetic Age Prediction from Blood

Pyrosequencing of the three age-associated CpGs was measured in 243 blood samples of healthy controls and 105 [17 (link)] and 80 [25 (link)] of these were also mentioned in previous work. Furthermore, we analyzed 70 independent AA and 18 independent DKC patients. Pyrosequencing was described in detail before [17 (link)] and performed at Cygenia GmbH (Aachen, Germany). In short, DNA was isolated from patient samples with the QIAamp DNA Mini and Blood Mini kit (Qiagen, Hilden, Germany) and bisulfite converted with the EZ DNA Methylation Kit (Zymo Research, Freiburg, Germany). Converted DNA was PCR amplified with the PyroMark PCR kit 800 (Qiagen) and 20 μL PCR product was immobilized to 60 μL Streptavidin Sepharose HP beads (GE Healthcare, Chicago, IL, USA), annealed to 0.8 μL 20 μM sequencing primers for 2 min at 80 °C (see all primer sequences in Additional file 1: Table S4). Samples were measured with the PyroMark Gold Q96 Reagents on a PyroMark Q96 ID and analyzed with the Pyro-Q-CpG 1.0.9 software (all from Qiagen). Epigenetic age was calculated as follows:
Predicted age (in years) = 38.0 − 26.4 × β value (cg02228185) − 23.7 × β value (cg25809905) + 164.7 × β value (CpG upstream of cg17861230)