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:Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser capture microdissection. Arabidopsis root cells undergoing giant cell formation due to nematode infestation and un-infested control root cells were laser captured and used to evaluate 2 amplification systems. One, NuGEN's WT-Ovation Pico amplification system, uses total RNA as starting material while the other, NuGEN's WT-One-Direct Amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The NuGEN WT-Ovation One-Direct system was less reproducible and more variable than the NuGEN WT-Ovation Pico system. The NuGEN WT-Ovation Pico Amplification kit resulted in the detection of thousands of genes differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the NuGEN WT-Ovation One-Direct Amplification kit. Evaluation of each amplification system consisted of three biological replicates per treatment (control root cells or giant cells) with one biological replicate split into three technical replicates for a total of 10 samples per amplification procedure. RNA samples for amplification with the NuGEN WT-Ovation Pico kit were isolated from 150 control cells or from 80 giant cells using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, USA). For amplification with the NuGEN WT-Ovation One-Direct kit, samples containing 150 control or 80 giant cells were collected directly into 2ul of the NuGEN lysis buffer. RNA amplifications were carried out according to the respective kitsâ?? protocols.

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

Dataset Information

Transcription profiling of mouse samples to compare three amplification methods for nanogram amounts of total RNA

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Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.

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