Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification and Correction of Amplification Protocol Bias in Microarray Studies

ABSTRACT: mRNA abundance microarrays are the most widely used high-throughput technology for functional genomics. But despite this extensive characterization, and despite their wide-spread adoption in discovery research, the uptake of microarrays as clinical diagnostic tools remains relatively limited due to sample degradation. Recently, the NuGEN WT-Ovation Pico kit was developed to resolve this problem. Here, we comprehensively evaluate its utility on two separate biological problems. First, we evaluate its effect on 12 malignant and 2 normal cervix samples. Second, we evaluate its effect on 2 normal kidney samples. In each case, we directly compare aliquots of the same RNA sample that are prepared by standard and low-input/low-RIN protocols to address three key questions. First, does the use of a low-input/degraded-sample protocol alter transcriptional profiles either globally or of specific genes? Second, do these changes alter biological conclusions drawn from the dataset? Third, are these changes biased towards specific classes of genes, either in terms of their sequence characteristics (e.g. length, GC-content) or their biological function? To achieve these goals, we selected 12 frozen cervix cancers and 2 unmatched normal samples. RNA was extracted and was split into two aliquots: one was prepared using the Nugen WT-Ovation Pico kit and the other using the standard Affymetrix Express array method. This approach allows us to directly assess the effect of sample preparation, in isolation of any other effects. These data were then pre-processed and the filtered genes were subject to linear-modeling to evaluate technical and biological variability. The resulting gene sets were then subject to pathway and sequence analysis, and LTR analysis. Lastly, to generalize these conclusions we repeated this experimental approach identically in two normal kidney samples and one RNA mixture control sample.

ORGANISM(S): Homo sapiens

SUBMITTER: Rui Yan

PROVIDER: E-GEOD-42764 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

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Identification and Correction of Amplification Protocol Bias in Microarray Studies

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