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Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots.

ABSTRACT: The Guthrie 903 card archived dried blood spots (DBSs) are a unique but terminal resource amenable for individual and population-wide genomic profiling. The limited amounts of DBS-derived genomic DNA (gDNA) can be whole genome amplified, producing sufficient gDNA for genomic applications, albeit with variable success; optimizing the isolation of high-quality DNA from these finite, low-yield specimens is essential. Agarose gel electrophoresis and spectrophotometry are established postextraction quality control (QC) methods but lack the power to disclose detailed structural, qualitative, or quantitative aspects that underlie gDNA failure in downstream applications. Visual automated fluorescence electrophoresis (VAFE) is a novel QC technology that affords precise quality, quantity, and molecular weight of double-stranded DNA from a single microliter of sample. We extracted DNA from 3-mm DBSs archived in the Swedish Neonatal Repository for >30 years and performed the first quantitative and qualitative analyses of DBS-derived DNA on VAFE, before and after whole genome amplified, in parallel with traditional QC methods. The VAFE QC data were correlated with subsequent sample performance in PCR, sequencing, and high-density comparative genome hybridization array. We observed improved standardization of nucleic acid quantity, quality and integrity, and high performance in the downstream genomic technologies. Addition of VAFE measures in QC increases confidence in the validity of genetic data and allows cost-effective downstream analysis of gDNA for investigational and diagnostic applications.

SUBMITTER: Klassen TL

PROVIDER: S-EPMC3644733 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots.

Klassen Tara L TL Drabek Janice J Tomson Torjbörn T Sveinsson Olafur O von Döbeln Ulrika U Noebels Jeffrey L JL Goldman Alicia M AM

The Journal of molecular diagnostics : JMD 20130319 3

The Guthrie 903 card archived dried blood spots (DBSs) are a unique but terminal resource amenable for individual and population-wide genomic profiling. The limited amounts of DBS-derived genomic DNA (gDNA) can be whole genome amplified, producing sufficient gDNA for genomic applications, albeit with variable success; optimizing the isolation of high-quality DNA from these finite, low-yield specimens is essential. Agarose gel electrophoresis and spectrophotometry are established postextraction q ...[more]

PMID: 23518217

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Project description:IntroductionHeel pricks are performed on newborns for diagnostic screenings of various pre-symptomatic metabolic and genetic diseases. Excess blood is spotted on Guthrie cards and archived by many states in biobanks for follow-up diagnoses and public health research. However, storage environment may vary across biobanks and across time within biobanks. With increased applications of DNA extracted from spots for genetic studies, identifying factors associated with genotyping success is critical to maximize DNA quality for future studies.MethodWe evaluated 399 blood spots, which were part of a genome-wide association study of childhood leukemia risk in children with Down syndrome, archived at the Michigan Neonatal Biobank between 1992 and 2008. High quality DNA was defined as having post-quality control call rate ≥ 99.0% based on the Illumina GenomeStudio 2.0 GenCall algorithm after processing the samples on the Illumina Infinium Global Screening Array. Bivariate analyses and multivariable logistic regression models were applied to evaluate effects of storage environment and storage duration on DNA genotyping quality.ResultsBoth storage environment and duration were associated with sample genotyping call rates (p-values < 0.001). Sample call rates were associated with storage duration independent of storage environment (p-trend = 0.006 for DBS archived in an uncontrolled environment and p-trend = 0.002 in a controlled environment). However, 95% of the total sample had high genotyping quality with a call rate ≥ 95.0%, a standard threshold for acceptable sample quality in many genetic studies.ConclusionBlood spot DNA quality was lower in samples archived in uncontrolled storage environments and for samples archived for longer durations. Still, regardless of storage environment or duration, neonatal biobanks including the Michigan Neonatal Biobanks can provide access to large collections of spots with DNA quality acceptable for most genotyping studies.

Project description:Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature.

Dataset Information

Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots.

Publications

Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots.

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