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Age-related micro-RNA abundance in individual C. elegans.

Lucanic M, Graham J, Scott G, Bhaumik D, Benz CC, Hubbard A, Lithgow GJ, Melov S - Aging (Albany NY) (2013)

Bottom Line: To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline.Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans.Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.

View Article: PubMed Central - PubMed

Affiliation: Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945, USA. mlucanic@buckinstitute.org

ABSTRACT
Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.

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Dynamic changes of miRNAs with age(A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1 and Day 12 of adulthood. (B-G) Normalized Ct values describing miRNA abundance in individual worms (each point represents data from a single animal) at two ages are shown for mir-44 (B), mir-51 (C), mir-240 (D), mir-58 (E), mir-60 (F), and mir-235 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs each point is the average from 2-3 technical replicates.
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Figure 1: Dynamic changes of miRNAs with age(A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1 and Day 12 of adulthood. (B-G) Normalized Ct values describing miRNA abundance in individual worms (each point represents data from a single animal) at two ages are shown for mir-44 (B), mir-51 (C), mir-240 (D), mir-58 (E), mir-60 (F), and mir-235 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs each point is the average from 2-3 technical replicates.

Mentions: To examine age-related miRNA expression changes we used the quantitative polymerase chain reaction (qPCR) to assay a panel of 69 miRNAs in RNA samples collected from 48 C. elegans animals. We used individual worms to assay the miRNA levels, an approach which has previously been used in the context of gene expression profiling via microarrays [5, 30]. Such an approach captures variation between animals with age, as it screens individuals as opposed to most conventional methods which analyze populations containing large numbers of pooled animals. Individual wild type (N2; Bristol strain) animals were examined for expression of each miRNA simultaneously using dynamic integrated nano-fluidic circuits (Fluidigm inc.) with Taqman qPCR assays (ABI) specific for each miRNA [31]. We first tested for expression of the miRNAs in 24 young (hermaphrodites on their first day of adulthood) and 24 old (hermaphrodites on their twelfth day of adulthood) animals. Young adult animals were non-gravid, while old animals were post-reproductive and were collected while the population was undergoing a high mortality rate (Figure 1A). We found that 45 miRNAs were consistently detected (in more than half of the animals) while 43 miRNAs were consistently detected in old animals (Supplemental Table 1). Of the 43 miRNAs that were consistently detected in both young and old animals, slightly more than half (56%) decreased in abundance with age, demonstrating that amongst the miRNAs we assayed, there is a trend towards most miRNAs decreasing in expression with age. As we had determined the levels of miRNAs in individual animals, we next examined the variation in abundance of each miRNA between individual worms for both young and old animals (standard deviation of the population from the mean Ct (cycle threshold)). There is a generalized increase in the variation of miRNA abundance with age, consistent with a generalized increase in stochastic dysregulation. Specifically we found that 68% of the miRNAs showed an increase in variation between individual worms with age, while if we only counted the miRNAs which were detected in all 48 animals then 74% of these showed an increase in their variation with age (Supplemental Table 1). This suggests that variability of miRNA expression generally increases with age. This increase in variability may be in part due to stochastic cell and tissue loss in aged animals [2, 32-34].


Age-related micro-RNA abundance in individual C. elegans.

Lucanic M, Graham J, Scott G, Bhaumik D, Benz CC, Hubbard A, Lithgow GJ, Melov S - Aging (Albany NY) (2013)

Dynamic changes of miRNAs with age(A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1 and Day 12 of adulthood. (B-G) Normalized Ct values describing miRNA abundance in individual worms (each point represents data from a single animal) at two ages are shown for mir-44 (B), mir-51 (C), mir-240 (D), mir-58 (E), mir-60 (F), and mir-235 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs each point is the average from 2-3 technical replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824409&req=5

Figure 1: Dynamic changes of miRNAs with age(A) Survivorship of a population of N2 (wild type) animals, from which individuals were harvested for analysis at Day 1 and Day 12 of adulthood. (B-G) Normalized Ct values describing miRNA abundance in individual worms (each point represents data from a single animal) at two ages are shown for mir-44 (B), mir-51 (C), mir-240 (D), mir-58 (E), mir-60 (F), and mir-235 (G). (H) Graphical representation of the fold change in miRNA abundance between young and old animals. In all graphs with error bars they indicate the standard error of the mean. In individual worm graphs each point is the average from 2-3 technical replicates.
Mentions: To examine age-related miRNA expression changes we used the quantitative polymerase chain reaction (qPCR) to assay a panel of 69 miRNAs in RNA samples collected from 48 C. elegans animals. We used individual worms to assay the miRNA levels, an approach which has previously been used in the context of gene expression profiling via microarrays [5, 30]. Such an approach captures variation between animals with age, as it screens individuals as opposed to most conventional methods which analyze populations containing large numbers of pooled animals. Individual wild type (N2; Bristol strain) animals were examined for expression of each miRNA simultaneously using dynamic integrated nano-fluidic circuits (Fluidigm inc.) with Taqman qPCR assays (ABI) specific for each miRNA [31]. We first tested for expression of the miRNAs in 24 young (hermaphrodites on their first day of adulthood) and 24 old (hermaphrodites on their twelfth day of adulthood) animals. Young adult animals were non-gravid, while old animals were post-reproductive and were collected while the population was undergoing a high mortality rate (Figure 1A). We found that 45 miRNAs were consistently detected (in more than half of the animals) while 43 miRNAs were consistently detected in old animals (Supplemental Table 1). Of the 43 miRNAs that were consistently detected in both young and old animals, slightly more than half (56%) decreased in abundance with age, demonstrating that amongst the miRNAs we assayed, there is a trend towards most miRNAs decreasing in expression with age. As we had determined the levels of miRNAs in individual animals, we next examined the variation in abundance of each miRNA between individual worms for both young and old animals (standard deviation of the population from the mean Ct (cycle threshold)). There is a generalized increase in the variation of miRNA abundance with age, consistent with a generalized increase in stochastic dysregulation. Specifically we found that 68% of the miRNAs showed an increase in variation between individual worms with age, while if we only counted the miRNAs which were detected in all 48 animals then 74% of these showed an increase in their variation with age (Supplemental Table 1). This suggests that variability of miRNA expression generally increases with age. This increase in variability may be in part due to stochastic cell and tissue loss in aged animals [2, 32-34].

Bottom Line: To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline.Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans.Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.

View Article: PubMed Central - PubMed

Affiliation: Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945, USA. mlucanic@buckinstitute.org

ABSTRACT
Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.

Show MeSH
Related in: MedlinePlus