Project description:Background: Standard Guthrie cards have been widely used to collect blood samples from neonates for newborn screening programs, and to a lesser extent, from normal controls and patients in research studies. Ease of blood collection (small quantity and less pain), transportation, and storage are the advantages of using these cards. It is believed that RNA obtained from these samples is of low quantity and degraded quality. However, we recently discovered that approximately 3,500 expressed genes can be detected from blood spot samples using in-house made, low resolution cDNA microarrays. Here, we established a new and improved methodology to acquire gene expression profiles from blood spot cards using commercially-available high resolution microarrays. We determined the optimal number of blood spot punches required for maximal RNA extraction, eliminated uses of trizol and chloroform for RNA extraction by using a modified protocol of the illustra Mini Spin Kit from GE-Whatm an, concentrated the quantity of RNA templates before amplification, improved amplification efficiency using the new Ribo-SPIA technology in WT-Ovation Pico System (WT-Pico) by NuGEN, before the samples were hybridized onto 4x44K whole human genome gene expression microarrays from Agilent. Nine dried blood spot samples were collected from a control population and stored at ~ -80 °C between 6 months to 2 years. High quality RNA was extracted from the buffy coat of the same individuals as a reference and processed using the standard Agilent microarray procedure. Commercially-available brain RNA was used as a positive control in both standard and new procedures for microarrays. Results: Three 3-mm punches produced the highest yield of total RNA using the non-trizol extraction method. Three to six nanogram per microliter of RNA can be concentrated and is sufficient to be amplified using the WT-Pico. Approximately 9,000 expressed genes can be detected after normalization and background correction of the microarray data. Conclusion: Genome-wide gene expression profile can be obtained from archived dried blood spot samples. Our new and improved methodology will add value to the perception of utilizing archival Guthrie cards eg. neonatal blood spot cards as unique biospecimens for molecular genomics and diagnostic studies of perinatal diseases such as pediatric cancers. Keywords: Gene Expression experiment

Project description:Background: Standard Guthrie cards have been widely used to collect blood samples from neonates for newborn screening programs, and to a lesser extent, from normal controls and patients in research studies. Ease of blood collection (small quantity and less pain), transportation, and storage are the advantages of using these cards. It is believed that RNA obtained from these samples is of low quantity and degraded quality. However, we recently discovered that approximately 3,500 expressed genes can be detected from blood spot samples using in-house made, low resolution cDNA microarrays. Here, we established a new and improved methodology to acquire gene expression profiles from blood spot cards using commercially-available high resolution microarrays. We determined the optimal number of blood spot punches required for maximal RNA extraction, eliminated uses of trizol and chloroform for RNA extraction by using a modified protocol of the illustra Mini Spin Kit from GE-Whatm an, concentrated the quantity of RNA templates before amplification, improved amplification efficiency using the new Ribo-SPIA technology in WT-Ovation Pico System (WT-Pico) by NuGEN, before the samples were hybridized onto 4x44K whole human genome gene expression microarrays from Agilent. Nine dried blood spot samples were collected from a control population and stored at ~ -80 °C between 6 months to 2 years. High quality RNA was extracted from the buffy coat of the same individuals as a reference and processed using the standard Agilent microarray procedure. Commercially-available brain RNA was used as a positive control in both standard and new procedures for microarrays. Results: Three 3-mm punches produced the highest yield of total RNA using the non-trizol extraction method. Three to six nanogram per microliter of RNA can be concentrated and is sufficient to be amplified using the WT-Pico. Approximately 9,000 expressed genes can be detected after normalization and background correction of the microarray data. Conclusion: Genome-wide gene expression profile can be obtained from archived dried blood spot samples. Our new and improved methodology will add value to the perception of utilizing archival Guthrie cards eg. neonatal blood spot cards as unique biospecimens for molecular genomics and diagnostic studies of perinatal diseases such as pediatric cancers. Keywords: Gene Expression experiment Archival guthrie blood-spot cards may contain valuable data for epidemiological or other studies. Showing microarray data from guthrie blood-spot cards

Project description:This Dataset goes alongside the 2023 manuscript: Novel perfluoroalkyl substances (PFAS) discovered in whole blood using automated non-targeted analysis of dried blood spots by Jeremy P. Koelmel, Elizabeth Z. Lin, Emily Parry, Paul Stelben, Emma E.Rennie, Krystal J.Godri Pollitt A small subset of per- and polyfluoroalkyl substances (PFAS) are routinely screened in human blood. These compounds generally explain <50 percent of the total PFAS in human blood. The percentage of known PFAS in human blood has been decreasing as replacement PFAS and more complex PFAS chemistries are introduced to the market. Most of these novel PFAS have not been previous identified. Non-targeted methods are required to characterize this dark matter PFAS. Our objective was to apply non-targeted PFAS analysis to human blood to gain an understanding about the sources, concentrations, and toxicity of these compounds. A high-resolution tandem mass spectrometry (HRMS) and software workflow for PFAS characterization in dried blood spots is reported. Dried blood spots are a less invasive collection technique compared to venous blood draws, allowing collection from vulnerable populations. Biorepositories of archived dried blood spots are available internationally from newborns and present opportunities to study prenatal exposure to PFAS. In this study, dried blood spot cards were analyzed using iterative MS/MS by liquid chromatography HRMS. Data processing was conducted using FluoroMatch Suite including a visualizer tool that presents homologous series, retention time vs m/z plots, MS/MS spectra, feature tables, annotations, and fragments for fragment screening. The researcher performing data-processing and annotation was blinded to the fact that standards were spiked in, and was able to annotated 95 percent of standards spiked on dried blood spot samples, signifying a low false negative rate using FluoroMatch Suite. A total of 28 PFAS (20 standards and 4 exogenous compounds) were detected across five homologous series with Schymanski Level 2 confidence. Of these four, 3 were perfluoroalkyl ether carboxylic acids (PFECA), a chemical class of PFAS which is increasingly being detected in environmental and biological matrices but is not currently screened in most targeted analyses. A further 86 potential PFAS were detected using fragment screening. PFAS are extremely persistent and widespread yet remain largely unregulated. Our findings will contribute to an improved an understanding of exposures. Application of these methods in environmental epidemiology studies have the potential to inform policy with regards to PFAS monitoring, regulation, and individual-level mitigation strategies.

Dataset Information

MicroRNA sequencing of BIBINS HIE dried blood spot

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