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Analysis of Dried Blood Spots by MALDI-MS

ABSTRACT: Concentrations of cocaine (5,3,2,1.5,1,0.75,0.5,0.25,0.125, 0.062, and 0.031 ppm) were spiked into whole human blood for MALDI-MS analysis. A 10 uL volume of blood/analyte mixture was allowed to dry on Whatman Grade 2 filter paper. A 1ppm concentration Cocaine-d3 was used as an internal standard. Parameters of the MALDI system were optimized, including laser energy, number of laser shots, and matrix. The internal standard was added on top of the dried blood spot sample. Wide isolation tandem MS was used to generate a calibration curve.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Blood

SUBMITTER: Elizabeth Dhummakupt

PROVIDER: ST000228 | MetabolomicsWorkbench | Thu Jul 16 00:00:00 BST 2015

REPOSITORIES: MetabolomicsWorkbench

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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

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Analysis of Dried Blood Spots by MALDI-MS

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