Unknown,Transcriptomics,Genomics,Proteomics

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Comparison between NuGEN's WT-Ovation Pico and One-Direct Amplification Systems

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Christopher Taylor

PROVIDER: E-GEOD-21981 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:affy_meloidogyne_rice - affy_meloidogyne_rice - Plant-parasitic nematodes cause profound economic losses to global agriculture with the obligate sedentary endoparasitic varieties; amongst them the cyst and Root Knot Nematode (RKN) species are the most damaging. Meloidogyne graminicola is a RKN mainly found in the monocotyledous plants. In the compatible interaction with Oryza sativa, M. graminicola induces the characteristic formation of hook-like galls resulting from the redifferentiation of root cells into multinucleate giant cells. In order to understand the global transcriptome changes occurring during infection, several recent microarray studies on root knots have demonstrated complex changes in host plant gene expression in response to infection. However, to our knowledge, all these transcriptome studies were performed on dicotyledous plants. A histological study enabled us to observe hyperplasia and hypertrophy of the surrounding cells leading to the formation of hook-like galls. We also investigated the plant response to M. graminicola by carrying out a global analysis of gene expression during gall formation in rice, using giant cell-enriched root tissues at an early stage (2dpi) and a latter stage (4dpi) of gall development.-Oryza sativa (var. Nipponbare) seedlings were grown on 6 cm3 SAP substrate completed with diluted Hoaglands solution (Reversat et al., 1999). Culture units were placed in a growth chamber illuminated with fluorescent tubes 9/24 h and maintained at 23°C for 5 days before being inoculated with a 100 J2-stage juveniles M. graminicola. One day after inoculation (dai), the rice seedlings were immersed in de-ionised water to remove all J2s that had not penetrated the roots and allowing synchronization of the infection. Each seedling was transferred to a hydroponic mini chamber (Reversat et al., 2004). Sampling was performed at 2 and 4 dai and each of them contained galls from 70 infected plants, they were then hand-dissected, frozen in liquid-nitrogen and stored at -80°C. As reference samples, uninfected meristematic root fragments were dissected from seedlings grown under the same conditions. Each sample was replicated 3 times. Keywords: normal vs disease comparison,time course 9 arrays - rice

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Comparison between NuGEN's WT-Ovation Pico and One-Direct Amplification Systems

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