Limits...
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
GAPDH mRNA and protein. (A) GAPDH mRNA between exon8 and exon9 were successfully amplified in two of the three nail RNA samples. The band of mRNA in nail 2 was brighter than in nail 3, but nail 1 failed to show an mRNA band on the gel. (B) Commessa staining of the nail protein extracts and immunoblotting for GAPDH protein. The amount of GAPDH in nail 2 was more than that of nail 3, and nail 1 failed to show the target protein band on the immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GAPDH mRNA and protein. (A) GAPDH mRNA between exon8 and exon9 were successfully amplified in two of the three nail RNA samples. The band of mRNA in nail 2 was brighter than in nail 3, but nail 1 failed to show an mRNA band on the gel. (B) Commessa staining of the nail protein extracts and immunoblotting for GAPDH protein. The amount of GAPDH in nail 2 was more than that of nail 3, and nail 1 failed to show the target protein band on the immunoblotting.

Mentions: Glyceraldehyde 3 phosphate dehydrogenase mRNA (113 bp of exon8-exon9) was successfully amplified from two samples (Figure 3A), and two samples of GAPDH protein showed positive bands in immunoblotting among three nails (Figure 3B). In nail 1, both GAPDH RNA and protein failed to show the bands on the gel or immunoblotting membrane. The amount of GAPDH RNA and protein in nail 2 showed thicker bands than those of nail 3 (Figure 3). From the same amount of RNA extracts and total protein, the expression profiles were quite different among the three subjects.


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)

GAPDH mRNA and protein. (A) GAPDH mRNA between exon8 and exon9 were successfully amplified in two of the three nail RNA samples. The band of mRNA in nail 2 was brighter than in nail 3, but nail 1 failed to show an mRNA band on the gel. (B) Commessa staining of the nail protein extracts and immunoblotting for GAPDH protein. The amount of GAPDH in nail 2 was more than that of nail 3, and nail 1 failed to show the target protein band on the immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GAPDH mRNA and protein. (A) GAPDH mRNA between exon8 and exon9 were successfully amplified in two of the three nail RNA samples. The band of mRNA in nail 2 was brighter than in nail 3, but nail 1 failed to show an mRNA band on the gel. (B) Commessa staining of the nail protein extracts and immunoblotting for GAPDH protein. The amount of GAPDH in nail 2 was more than that of nail 3, and nail 1 failed to show the target protein band on the immunoblotting.
Mentions: Glyceraldehyde 3 phosphate dehydrogenase mRNA (113 bp of exon8-exon9) was successfully amplified from two samples (Figure 3A), and two samples of GAPDH protein showed positive bands in immunoblotting among three nails (Figure 3B). In nail 1, both GAPDH RNA and protein failed to show the bands on the gel or immunoblotting membrane. The amount of GAPDH RNA and protein in nail 2 showed thicker bands than those of nail 3 (Figure 3). From the same amount of RNA extracts and total protein, the expression profiles were quite different among the three subjects.

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