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Nail DNA and possible biomarkers: a pilot study.

Park J, Liang D, Kim JW, Luo Y, Huang T, Kim SY, Chang SS - J Prev Med Public Health (2012)

Bottom Line: Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts.Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study.Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

View Article: PubMed Central - PubMed

Affiliation: Genetic Epidemiology Research Institute, University of California, Irvine, USA.

ABSTRACT

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples.

Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments.

Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study.

Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

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Family pedigree and comparison of target amplification among 12 subjects. (A) Each family has three generations composed of the grandparents (69 to 74 years old), parents (43 to 46 years old) and two children (7 to 11 years old). The mothers in the two families were daughters of the same parents. (B) The threshold cycles of three different amplifications among the two children and 400 bp amplification of the grandmother in family 2 showed significantly delayed performance in amplification (ANOVA/Duncan Scheffe among the twelve subjects). Family 2 in total showed significantly delayed threshold cycles in amplification of all three segments compared to those of family 1 in total (t-test between the families). (C) The difference in the threshold cycles between the longer and shorter segments showed a larger standard deviation than the threshold cycles among twelve subjects. Among the 12 subjects, the grandmother of the family 1 showed significantly greater difference in threshold cycles than the other subjects (ANOVA/Duncan Scheffe). Family 2 in total showed a greater difference in both ratios of the two sizes comparison than those of family 1 in total (t-test between the families). mt-Co 1, mitochondrially encoded cytochrome C oxidase 1.
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Figure 1: Family pedigree and comparison of target amplification among 12 subjects. (A) Each family has three generations composed of the grandparents (69 to 74 years old), parents (43 to 46 years old) and two children (7 to 11 years old). The mothers in the two families were daughters of the same parents. (B) The threshold cycles of three different amplifications among the two children and 400 bp amplification of the grandmother in family 2 showed significantly delayed performance in amplification (ANOVA/Duncan Scheffe among the twelve subjects). Family 2 in total showed significantly delayed threshold cycles in amplification of all three segments compared to those of family 1 in total (t-test between the families). (C) The difference in the threshold cycles between the longer and shorter segments showed a larger standard deviation than the threshold cycles among twelve subjects. Among the 12 subjects, the grandmother of the family 1 showed significantly greater difference in threshold cycles than the other subjects (ANOVA/Duncan Scheffe). Family 2 in total showed a greater difference in both ratios of the two sizes comparison than those of family 1 in total (t-test between the families). mt-Co 1, mitochondrially encoded cytochrome C oxidase 1.

Mentions: In the feasibility study of nail DNA for monitoring an individual's health status, the integrity and quantity of DNA were analyzed using nail clippings from 12 volunteer subjects from two related families, under institutional review board study protocol (UCI IRB HS#2008-1697). Three age groups-4 participants each in their teens, forties, and seventies-were recruited as shown Figure 1A. They were related, to minimize genetic and environmental variation and to give more power to detect possible differences in other factors such as age, sex, or among collections. The subjects voluntarily donated their fingernail and toenail clippings three times at one-month interval. The individuals were in homeostatic healthy status for a year before and during the study period. The collected samples were stored at room temperature until the extraction of biomolecules.


Nail DNA and possible biomarkers: a pilot study.

Park J, Liang D, Kim JW, Luo Y, Huang T, Kim SY, Chang SS - J Prev Med Public Health (2012)

Family pedigree and comparison of target amplification among 12 subjects. (A) Each family has three generations composed of the grandparents (69 to 74 years old), parents (43 to 46 years old) and two children (7 to 11 years old). The mothers in the two families were daughters of the same parents. (B) The threshold cycles of three different amplifications among the two children and 400 bp amplification of the grandmother in family 2 showed significantly delayed performance in amplification (ANOVA/Duncan Scheffe among the twelve subjects). Family 2 in total showed significantly delayed threshold cycles in amplification of all three segments compared to those of family 1 in total (t-test between the families). (C) The difference in the threshold cycles between the longer and shorter segments showed a larger standard deviation than the threshold cycles among twelve subjects. Among the 12 subjects, the grandmother of the family 1 showed significantly greater difference in threshold cycles than the other subjects (ANOVA/Duncan Scheffe). Family 2 in total showed a greater difference in both ratios of the two sizes comparison than those of family 1 in total (t-test between the families). mt-Co 1, mitochondrially encoded cytochrome C oxidase 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3412986&req=5

Figure 1: Family pedigree and comparison of target amplification among 12 subjects. (A) Each family has three generations composed of the grandparents (69 to 74 years old), parents (43 to 46 years old) and two children (7 to 11 years old). The mothers in the two families were daughters of the same parents. (B) The threshold cycles of three different amplifications among the two children and 400 bp amplification of the grandmother in family 2 showed significantly delayed performance in amplification (ANOVA/Duncan Scheffe among the twelve subjects). Family 2 in total showed significantly delayed threshold cycles in amplification of all three segments compared to those of family 1 in total (t-test between the families). (C) The difference in the threshold cycles between the longer and shorter segments showed a larger standard deviation than the threshold cycles among twelve subjects. Among the 12 subjects, the grandmother of the family 1 showed significantly greater difference in threshold cycles than the other subjects (ANOVA/Duncan Scheffe). Family 2 in total showed a greater difference in both ratios of the two sizes comparison than those of family 1 in total (t-test between the families). mt-Co 1, mitochondrially encoded cytochrome C oxidase 1.
Mentions: In the feasibility study of nail DNA for monitoring an individual's health status, the integrity and quantity of DNA were analyzed using nail clippings from 12 volunteer subjects from two related families, under institutional review board study protocol (UCI IRB HS#2008-1697). Three age groups-4 participants each in their teens, forties, and seventies-were recruited as shown Figure 1A. They were related, to minimize genetic and environmental variation and to give more power to detect possible differences in other factors such as age, sex, or among collections. The subjects voluntarily donated their fingernail and toenail clippings three times at one-month interval. The individuals were in homeostatic healthy status for a year before and during the study period. The collected samples were stored at room temperature until the extraction of biomolecules.

Bottom Line: Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts.Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study.Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

View Article: PubMed Central - PubMed

Affiliation: Genetic Epidemiology Research Institute, University of California, Irvine, USA.

ABSTRACT

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples.

Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments.

Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study.

Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

Show MeSH