The science behind the test

What is Aging?

Aging is a universal biological process that manifests in general decline in our health and vitality that leads to the high probability of death. One of the worst parts of aging is the decline in several body functions, most notably, aa reduced mass and contraction power of the muscle (known as sarcopenia), decreased bone density, deterioration of the cardiovascular system, decline in cognitive functions and a general pro-inflammatory state of the whole organism. This decline represents the primary risk factor for the development of various high debilitating age-related diseases, such as cancer, Alzheimer’s, diabetes, etc.

The fantastic innovations and discoveries in medicine have led to a significant increase in human lifespan. According to the data from World Population Prospects [ 2015 Revision (UN, 2015)], the human population will reach 10 billion by 2100, and 22.3% of the whole population will be aged 65 or over. In fact, in 2015, one in eight people worldwide was aged 60 years or over. By 2030, older persons are projected to account for one in six people globally and will outnumber children aged 0-9 years. By the middle of the twenty-first century, one in every five people will be aged 60 years or over, and there will be more people aged 60 years or above than adolescents and youth aged 10-24 years. This is a huge problem, as aging comes with not only wisdom but with a high probability of development of age-related diseases and disabilities. An increase in aging population means an increase in health care costs and the decrease in workforce.

One can easily assume that if we manage to slow down aging, we can prevent age-related diseases and live longer and happier lives. Naturally, to control the aging process, we need to learn how to measure the rate of aging in real time. Aging is usually measured chronologically (in days or years). However, aging almost never perfectly correlated with time. Our exact biological age is influenced by many additional factors, such as genetic background lifestyle, and diseases.

To address this challenge, several biological markers of aging have been developed. According to to the American Federation for Aging Reseach (AFAR), these markers have to answer to the following criteria: (1) It must predict the rate of aging. In other words, it would tell precisely where a person is in their total lifespan. It must be a better predictor of lifespan than chronological age; (2) It must monitor a primary process that underlies the aging process, not the effects of the disease; (3) It must be able to be repeatedly tested without harming the person; (4) It must be something that works in humans and in laboratory animals, such as mice. This is so that it can be tested in lab animals before being validated in humans. These markers are unique molecules that can be separated into molecular- (based on DNA, RNA, etc.) or phenotypic biomarkers of aging (clinical measures such as blood pressure, grip strength, lipids, etc.).

Why test with saliva?

Why do we use saliva to determine your true biological age



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What can our DNA tell us about our age?

What is in your genes?

Every human is unique, and this uniqueness is mainly guided by a set of approximately 25,000 genes. Genes are words that are built in the sentences that guide our body through life. These genes determine our appearances, such as the color of our eyes, h, ir and skin, height, etc. They also guide the function of all organs and tissues in our body. If mistakes (mutations: changes in words and sentences grammar, deletion, or duplication) in genes occur, they could lead to disorganization if your bodily functions. For example, we are lactose intolerant, very sensitive to bitter taste, or do not have a very athletic muscle composition. Although this information is extremely useful to define your personalized healthy lifestyle, the genetic profiling would not show if your anti-aging program is working. These traits are written in a hard copy of our genetic material and cannot be reversed. There are many companies that offer the genetic test to determine your genetic composition, as well as ancestry and wellness reports that could be derived from your unique genetic code.

Is Telomere Length a Biomarker of Aging?

The quest for the more dynamic biomarker for aging lead to the discovery of the role of telomeres in the aging process and as an aging biomarker. Telomeres are the protective caps on the ends of the strands of DNA called chromosomes, which house our genomes. In young humans, telomeres are about 8,000-10,000 nucleotides long, and with every cell division, they get shorter. On average, a healthy individual decreases its telomere length at a rate of 24.8–27.7 base pairs per year. When the length of the telomere reaches a critical length the cell stops dividing and dies. This internal “clock” is accelerated by various parameters, such as obesity, smoking, lack of physical activity, stress, exposure to pollution, etc. It was proposed that good diet (high fiber, plenty of antioxidants, lean/low protein), and regular exercise can potentially reduce the rate of telomere shortening, disease risk, and pace of aging. It is clear that telomeres are involved in cellular aging and human diseases of premature aging (for example, Hutchinson-Gilford syndrome and Werner syndrome). However, it is still inconclusive whether telomere length is a biomarker of aging. Currently, telomere length does not fully meet American Federation of Aging Research criteria that telomere length is (a) a better predictor of lifespan than chronological age and that (b) it monitors a basic process underlying normal aging at the population level. It should be noted, that the use of blood samples is only valid if telomere length estimated. Whether telomere length measured in peripheral leukocytes is a surrogate marker for other tissues requires more investigation.

Epigenetic clock?

In addition to mutations, our genes activity is also modulated by chemical modifications. In the recent years, one particular modification received a special attention in the scientific community due to its ability to accurately predict biological age. This modification called DNA methylation, a process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. Instead, DNA methyl groups attract proteins (histones) that cover DNA ensure its stability and proper gene expression, making DNA methylation is vital to healthy growth and development. This process called epigenetic regulation of gene expression. DNA methylation naturally occurs in all our cells and the level of methylations highly correlated with our behavior, lifestyle choices, and biological age. Most importantly, in contrast to DNA mutations, epigenetic alterations are reversible and as such are promising targets for devising therapeutic approaches aimed at slowing the inevitable process of aging. The genetic test that determines the level of the landscape of DNA methylation is called epigenetic analysis.

Several studies in the recent years identified a measure of DNA methylation as a viable age predictor (also referred as the Epigenetic clock). The epigenetic clock was developed on large (thousands) samples covering the entire adult lifespan and different ethnic populations. The most striking feature of the epigenetic clocks is their ability to predict all-cause mortality. DNA methylation naturally occurs in all our cells and the level of methylations highly correlated with our behavior, lifestyle choices, aging phenotypes (frailty, poorer cognitive performance, muscle strength, etc), age-related diseases (Alzheimer’s, cancer, etc), and biological age.

Until recently, epigenetic testing was conducted only by research institutions due to high costs and very invasive sample collection procedure. TruMe developed first ever direct to consumer epigenetic test – TruAge Index-that calculates the biological age of individuals by analyzing the DNA methylation (DNAm) profiles of several loci in the human genome as a biomarker of the aging process. TruAge Index has the super easy sample collection procedure. Now anyone can submit their DNA for analysis to determine how old they really are and receive a recommendation on how to reverse or slow down aging. The TruAge Index is highly reflective of age-related changes in metabolism, physiology, and most importantly – lifestyle. During the design of TAI we focused on the following aspects: (1) Our test should be easily adapted to self-sampling protocol; (2) Accurately predict true biological age with the minimum margin of error (small MAD ≤ 5 years (MAD = mean absolute deviation)); (3) The testing method should be cost-effective; (4) The margin of error for subsequent samples should be no more than than two month.

A large number of recent studies provided information on age-related methylation sites that either hypermethylated (almost every cysteine residue in the target loci is methylated) or hypomethylated (methylation at the target loci is lost) during the aging process in different tissue samples (see references below: 1–14). Clearly, the friendliest tissue for the self-sampling home-based test is saliva or buccal swabs. We pooled publicly available DNAm profiles derived from saliva/buccal swabs samples of healthy individuals that can best predict donor age. Overall we analyzed over 900 samples for saliva (n=800 9, 6, 3,10,11) and for buccal swabs ( (n=109)12). We obtained a small set of 13 methylation markers that showed a high correlation between DNA methylation pattern and age. We used bioinformatics to combine these markers in 4 groups that individually demonstrated the high correlation between predicted and chronological age, with MAD from chronological age ranges from 3.4-5.5 years. Notably, these DNA methylation patterns were also highly predictive of chronological age in blood samples and several other tissues.

How do we perform our test?

We isolate white blood cells from the saliva samples. We break cells open and isolate total DNA. We treat your DNA with sodium bisulfite to convert all unmethylated cysteine residues to the uracil (see Figure 1 on the right for the detailed explanation of the method). During subsequent PCRs, uracil residues are replicated as thymine residues, and methylated cytosine residues are replicated as cytosines Thus, the bisulfite-treated DNA is used as a template to amplify specific loci using methylation-specific PCR (polymerase chain reaction) primers.


The PCR-based methods that use sodium bisulfite-treated DNA as a template is generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. The sequence analysis surrounding most highly predictive methylation sites were selected for locus-specific DNAm analysis by Sanger sequencing or pyrosequencing. We quantify the percent of methylated and unmethylated cysteine residues at each locus. These values are used in our proprietary bioinformatic pipeline to calculate your true biological age.
For validation, we have used an independent set of samples (thank you very much to all who participated in this study!!!! True friends). We have used the most stringent validation possible, and the results were highly reproducible. We analyzed the biological age of 50 individuals using all 13 highly predictive genomic loci. Our data demonstrate reasonable precision in predictions of chronological age (Figure 2). We expect our model to improve significantly on either a much more significant number of samples or by simultaneous measurement additional age-associated DNAm markers.


For more on Epigenetics:



  1. Hannum, G. et al. Genome-wide Methylation Profiles Reveal Quantitative Views of Human Aging Rates. Mol. Cell 49, 359–367 (2012).
  2. Horvath Genome, H. & Horvath, S. DNA methylation age of human tissues and cell types. (2013). at <>
  3. Bocklandt, S. et al. Epigenetic Predictor of Age. (2011). doi:10.1371/
  4. Koch, C. M. & Wagner, W. Epigenetic-aging-signature to determine age in different tissues. Aging (Albany. NY). 3, 1018–27 (2011).
  5. Eipel, M. et al. Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures. Aging (Albany. NY). 8, 1034–1048 (2017).
  6. Hong, S. R. et al. DNA methylation-based age prediction from saliva: High age predictability by combination of 7 CpG markers. Forensic Sci. Int. Genet. 29, 118–125 (2017).
  7. Miyaki K et al. DNA Methylation Status of the Methylenetetrahydrofolate Reductase Gene is associated with Depressive Symptoms in Japanese Workers: A Cross-Sectional Study. at <>
  8. Bacalini, M. G. et al. Systemic Age-Associated DNA Hypermethylation of ELOVL2 Gene: In Vivo and in Vitro Evidences of a Cell Replication Process. Journals Gerontol. – Ser. A Biol. Sci. Med. Sci. 72, 1015–1023 (2017).
  9. Horvath, S. et al. An epigenetic clock analysis of race/ethnicity, sex, and coronary heart disease An epigenetic clock analysis of race/ ethnicity, sex, and coronary heart disease. Genome Biol. 17, (2016).
  10. Liu, J., Morgan, M., Hutchison, K. & Calhoun, V. D. A Study of the Influence of Sex on Genome Wide Methylation. PLoS One 5, (2010).
  11. Yp Souren, N. et al. Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles. Genome Biol. 14, R44 (2013).
  12. Essex, M. J. et al. Epigenetic Vestiges of Early Developmental Adversity: Childhood Stress Exposure and DNA Methylation in Adolescence. doi:10.1111/j.1467-8624.2011.01641.x
  13. Kuhn, M. Building Predictive Models in R Using the caret Package. J. Stat. Softw. 28, 1–26 (2008).
  14. Leakey, T. I. et al. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms. Nucleic Acids Res. 36, (2008).