Fig. 1: Methodology overview, model overlap, and heat map of significant changes in RNA (ENSG) levels.

a Flowchart of sample preparation (orange) and analytical methods (green to gray) used in this study. b Volcano plots capture all significant (p < 0.01 from two-sided Wald test, unadjusted) RNAs identified by our models: linear model including all RNAs, standard negative binomial model including only RNAs with all non-zero CPB values, and zero-adjusted negative binomial model including only RNAs with at least one value of zero CPB. Red and blue points indicate significant RNAs with positive and negative β-values for age, respectively. Black points denote RNAs with nonsignificant β-values for age. c Venn diagram displaying the overlap between all RNAs significantly associated with aging identified by the linear (blue) and negative binomial (yellow) models (overlap shown in brown). Black denotes total significant RNAs, whereas red and blue values represent significant RNAs with positive and negative β-values for age, respectively. d Heat map representing the expression levels of the 506 significant RNAs shared between the linear and negative binomial models with age. Row-wise z-scores were calculated and color-coded to display changes in RNA expression with increasing age (22–83 years old). Light yellow and dark blue bars indicate RNAs with very low and very high expression levels, respectively, at a particular age. In addition, a correlation-based distance method was used to cluster the RNAs obtained from the linear model with positive and negative β-values for age. RNAs with positive β-values are shown in the top of the heat map, containing RNAs with low to high expression (yellow to blue rows) with increasing age. Similarly, RNAs with negative β-values are shown on the bottom of the heat map, containing RNAs with high to low expression (blue to yellow rows) with increasing age.