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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.

Show MeSH
Nested target amplification of mitochondrial DNA (mtDNA). (A) Over 9.2 kb was successfully amplified in three nail DNA samples and the blood(+) control. Nested polymerase chain reaction for mtDNA segments for 3121 to 3319 (B,D) and 8825 to 9090 (C,E) were also successfully quantified in three nail DNA samples and the blood sample.
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Figure 2: Nested target amplification of mitochondrial DNA (mtDNA). (A) Over 9.2 kb was successfully amplified in three nail DNA samples and the blood(+) control. Nested polymerase chain reaction for mtDNA segments for 3121 to 3319 (B,D) and 8825 to 9090 (C,E) were also successfully quantified in three nail DNA samples and the blood sample.

Mentions: From three samples of nail DNA, 9.251 kb (mt569 to mt9819) was successfully amplified (Figure 2). Using 1 µL of product, downstream nest qPCR was performed. The amount of DNA used for qPCR were ranged from 143.6 to 150.0 ng in 1 µL of nail DNA.


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)

Nested target amplification of mitochondrial DNA (mtDNA). (A) Over 9.2 kb was successfully amplified in three nail DNA samples and the blood(+) control. Nested polymerase chain reaction for mtDNA segments for 3121 to 3319 (B,D) and 8825 to 9090 (C,E) were also successfully quantified in three nail DNA samples and the blood sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Nested target amplification of mitochondrial DNA (mtDNA). (A) Over 9.2 kb was successfully amplified in three nail DNA samples and the blood(+) control. Nested polymerase chain reaction for mtDNA segments for 3121 to 3319 (B,D) and 8825 to 9090 (C,E) were also successfully quantified in three nail DNA samples and the blood sample.
Mentions: From three samples of nail DNA, 9.251 kb (mt569 to mt9819) was successfully amplified (Figure 2). Using 1 µL of product, downstream nest qPCR was performed. The amount of DNA used for qPCR were ranged from 143.6 to 150.0 ng in 1 µL of nail DNA.

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