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Microarray Based Comparison of three Amplification Methods For Nanogram Amounts of Total RNA

ABSTRACT: Two T7 based methods One round of Amplification (Affymetrix) and Two round of Amplification were compared to two Ribo-SPIA based systems, RiboSPIA and pico Ribo SPIA systems. Data for Pico-RiboSPIA are listed here. All Hybridisation were performed using Affymetrix Mouse 430-2 gene chips. Data were all scaled to 500. For 12 chips performed with pico Ribo SPIA the scaling factor average was 2.0 +/- 0.3, background intensities 74.4 +/- 12.7 , noise 3.9 +/- 0.6, rawQ 2.5 +/- 0.3 Keywords: ordered

ORGANISM(S): Mus musculus

PROVIDER: GSE2019 | GEO | 2004/12/21

SECONDARY ACCESSION(S): PRJNA91841

REPOSITORIES: GEO

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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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Microarray Based Comparison of three Amplification Methods For Nanogram Amounts of Total RNA

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