Nail DNA and Possible Biomarkers: A Pilot Study

Abstract

ObjectivesNail 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.MethodsThree 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.ResultsThe 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.ConclusionsNail 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.

Fast skeletal muscle transcriptome of the Gilthead sea bream (Sparus aurata) determined by next generation sequencing

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DNA from Nails for Genetic Analyses in Large-Scale Epidemiologic Studies

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“…Diluted NaOH was added to redissolve the precipitates. Next, an equal volume of phenol/chloroform was added to the neutralized sample, mixed for 10 minutes by inversion, and centrifuged at 19,000 Âg for 15 minutes at 4 C. The aqueous top layer was transferred to a clean tube and DNA was precipitated by adding 1/10 volume (35 mL) of 3 mol/L sodium acetate (pH, 5.2), 2 volumes (700 mL) of 95% ethanol and 1 mL 20 mg/mL glycogen overnight at À20 C. DNA was pelleted by centrifugation at 19,000 Âg for 30 minutes at 4 C, washed once in 700 mL 75% ethanol, centrifuged at 19,000 Âg for 15 minutes at 4 C, after which the supernatant was removed and the pellet dried and resuspended in 100 mL 10 mmol/L Tris-HCl (pH, 8).…”

Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method

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