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Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray.

ABSTRACT: BACKGROUND:Microarrays are a powerful tool used for the determination of global RNA expression. There is an increasing requirement to focus on profiling gene expression in tissues where it is difficult to obtain large quantities of material, for example individual tissues within organs such as the root, or individual isolated cells. From such samples, it is difficult to produce the amount of RNA required for labelling and hybridisation in microarray experiments, thus a process of amplification is usually adopted. Despite the increasing use of two-cycle amplification for transcriptomic analyses on the Affymetrix ATH1 array, there has been no report investigating any potential bias in gene representation that may occur as a result. RESULTS:Here we compare transcriptomic data generated using Affymetrix one-cycle (standard labelling protocol), two-cycle (small-sample protocol) and IVT-Express protocols with the Affymetrix ATH1 array using Arabidopsis root samples. Results obtained with each protocol are broadly similar. However, we show that there are 35 probe sets (of a total of 22810) that are misrepresented in the two-cycle data sets. Of these, 33 probe sets were classed as mis-amplified when comparisons of two independent publicly available data sets were undertaken. CONCLUSIONS:Given the unreliable nature of the highlighted probes, we caution against using data associated with the corresponding genes in analyses involving transcriptomic data generated with two-cycle amplification protocols. We have shown that the Affymetrix IVT-E labelling protocol produces data with less associated bias than the two-cycle protocol, and as such, would recommend this kit for new experiments that involve small samples.

SUBMITTER: Holman TJ

PROVIDER: S-EPMC2847557 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray.

Holman Tara J TJ Wilson Michael H MH Kenobi Kim K Dryden Ian L IL Hodgman T Charlie TC Wood Andrew Ta AT Holdsworth Michael J MJ

Plant methods 20100315

<h4>Background</h4>Microarrays are a powerful tool used for the determination of global RNA expression. There is an increasing requirement to focus on profiling gene expression in tissues where it is difficult to obtain large quantities of material, for example individual tissues within organs such as the root, or individual isolated cells. From such samples, it is difficult to produce the amount of RNA required for labelling and hybridisation in microarray experiments, thus a process of amplifi ...[more]

PMID: 20230623

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Project description:BackgroundHigh throughput parallel sequencing, RNA-Seq, has recently emerged as an appealing alternative to microarray in identifying differentially expressed genes (DEG) between biological groups. However, there still exists considerable discrepancy on gene expression measurements and DEG results between the two platforms. The objective of this study was to compare parallel paired-end RNA-Seq and microarray data generated on 5-azadeoxy-cytidine (5-Aza) treated HT-29 colon cancer cells with an additional simulation study.MethodsWe first performed general correlation analysis comparing gene expression profiles on both platforms. An Errors-In-Variables (EIV) regression model was subsequently applied to assess proportional and fixed biases between the two technologies. Then several existing algorithms, designed for DEG identification in RNA-Seq and microarray data, were applied to compare the cross-platform overlaps with respect to DEG lists, which were further validated using qRT-PCR assays on selected genes. Functional analyses were subsequently conducted using Ingenuity Pathway Analysis (IPA).ResultsPearson and Spearman correlation coefficients between the RNA-Seq and microarray data each exceeded 0.80, with 66%~68% overlap of genes on both platforms. The EIV regression model indicated the existence of both fixed and proportional biases between the two platforms. The DESeq and baySeq algorithms (RNA-Seq) and the SAM and eBayes algorithms (microarray) achieved the highest cross-platform overlap rate in DEG results from both experimental and simulated datasets. DESeq method exhibited a better control on the false discovery rate than baySeq on the simulated dataset although it performed slightly inferior to baySeq in the sensitivity test. RNA-Seq and qRT-PCR, but not microarray data, confirmed the expected reversal of SPARC gene suppression after treating HT-29 cells with 5-Aza. Thirty-three IPA canonical pathways were identified by both microarray and RNA-Seq data, 152 pathways by RNA-Seq data only, and none by microarray data only.ConclusionsThese results suggest that RNA-Seq has advantages over microarray in identification of DEGs with the most consistent results generated from DESeq and SAM methods. The EIV regression model reveals both fixed and proportional biases between RNA-Seq and microarray. This may explain in part the lower cross-platform overlap in DEG lists compared to those in detectable genes.

Dataset Information

Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray.

Publications

Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray.

Similar Datasets

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