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Microarrays, deep sequencing and the true measure of the transcriptome.

Malone JH, Oliver B - BMC Biol. (2011)

Bottom Line: Microarrays first made the analysis of the transcriptome possible, and have produced much important information.Today, however, researchers are increasingly turning to direct high-throughput sequencing -- RNA-Seq -- which has considerable advantages for examining transcriptome fine structure -- for example in the detection of allele-specific expression and splice junctions.We conclude that microarrays remain useful and accurate tools for measuring expression levels, and RNA-Seq complements and extends microarray measurements.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Developmental Biology, National Institute of Digestive, Diabetes, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. malonej@niddk.nih.gov

ABSTRACT
Microarrays first made the analysis of the transcriptome possible, and have produced much important information. Today, however, researchers are increasingly turning to direct high-throughput sequencing -- RNA-Seq -- which has considerable advantages for examining transcriptome fine structure -- for example in the detection of allele-specific expression and splice junctions. In this article, we discuss the relative merits of the two techniques, the inherent biases in each, and whether all of the vast body of array work needs to be revisited using the newer technology. We conclude that microarrays remain useful and accurate tools for measuring expression levels, and RNA-Seq complements and extends microarray measurements.

Show MeSH
Data production workflow for microarrays. Microarrays require labeling of target material, hybridization to arrays, washing, and scanning to obtain measures of gene expression. RNA converted to cDNA from the sample will hybridize to the corresponding oligonucleotide targets, so that more highly expressed genes will be reflected in more abundant material hybridized and thus greater fluorescence intensity. In modern arrays, multiple probes are designed for a single gene in order to obtain fluorescence intensities that can be used as an index of gene expression.
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Figure 1: Data production workflow for microarrays. Microarrays require labeling of target material, hybridization to arrays, washing, and scanning to obtain measures of gene expression. RNA converted to cDNA from the sample will hybridize to the corresponding oligonucleotide targets, so that more highly expressed genes will be reflected in more abundant material hybridized and thus greater fluorescence intensity. In modern arrays, multiple probes are designed for a single gene in order to obtain fluorescence intensities that can be used as an index of gene expression.

Mentions: Figure 1 describes an expression experiment designed to identify genes that are differentially expressed in D. pseudoobscura female and male heads, which were manually dissected from flies over dry ice, after which total RNA was extracted followed by a poly A+ selection. poly A+ selected mRNA was converted to cDNA using end-labeled random nonamers and reverse transcription. During this reaction, a fluorophore is added to the 5' end of each short cDNA. In this case, the cDNA of one sex was labeled with one type of fluorescent dye (cyanine 3 or Cy3) and the cDNA of the other sex was labeled with a different fluorescent dye (cyanine 5 or Cy5) with fluorescence at a different wavelength. We generated replicate samples (N = 4) and samples with dyes swapped between females and males in order to control for technical artifacts due to labeling and dye biases and to measure the inherent variability in gene expression irrespective of the sex of the sample.


Microarrays, deep sequencing and the true measure of the transcriptome.

Malone JH, Oliver B - BMC Biol. (2011)

Data production workflow for microarrays. Microarrays require labeling of target material, hybridization to arrays, washing, and scanning to obtain measures of gene expression. RNA converted to cDNA from the sample will hybridize to the corresponding oligonucleotide targets, so that more highly expressed genes will be reflected in more abundant material hybridized and thus greater fluorescence intensity. In modern arrays, multiple probes are designed for a single gene in order to obtain fluorescence intensities that can be used as an index of gene expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104486&req=5

Figure 1: Data production workflow for microarrays. Microarrays require labeling of target material, hybridization to arrays, washing, and scanning to obtain measures of gene expression. RNA converted to cDNA from the sample will hybridize to the corresponding oligonucleotide targets, so that more highly expressed genes will be reflected in more abundant material hybridized and thus greater fluorescence intensity. In modern arrays, multiple probes are designed for a single gene in order to obtain fluorescence intensities that can be used as an index of gene expression.
Mentions: Figure 1 describes an expression experiment designed to identify genes that are differentially expressed in D. pseudoobscura female and male heads, which were manually dissected from flies over dry ice, after which total RNA was extracted followed by a poly A+ selection. poly A+ selected mRNA was converted to cDNA using end-labeled random nonamers and reverse transcription. During this reaction, a fluorophore is added to the 5' end of each short cDNA. In this case, the cDNA of one sex was labeled with one type of fluorescent dye (cyanine 3 or Cy3) and the cDNA of the other sex was labeled with a different fluorescent dye (cyanine 5 or Cy5) with fluorescence at a different wavelength. We generated replicate samples (N = 4) and samples with dyes swapped between females and males in order to control for technical artifacts due to labeling and dye biases and to measure the inherent variability in gene expression irrespective of the sex of the sample.

Bottom Line: Microarrays first made the analysis of the transcriptome possible, and have produced much important information.Today, however, researchers are increasingly turning to direct high-throughput sequencing -- RNA-Seq -- which has considerable advantages for examining transcriptome fine structure -- for example in the detection of allele-specific expression and splice junctions.We conclude that microarrays remain useful and accurate tools for measuring expression levels, and RNA-Seq complements and extends microarray measurements.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Developmental Biology, National Institute of Digestive, Diabetes, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. malonej@niddk.nih.gov

ABSTRACT
Microarrays first made the analysis of the transcriptome possible, and have produced much important information. Today, however, researchers are increasingly turning to direct high-throughput sequencing -- RNA-Seq -- which has considerable advantages for examining transcriptome fine structure -- for example in the detection of allele-specific expression and splice junctions. In this article, we discuss the relative merits of the two techniques, the inherent biases in each, and whether all of the vast body of array work needs to be revisited using the newer technology. We conclude that microarrays remain useful and accurate tools for measuring expression levels, and RNA-Seq complements and extends microarray measurements.

Show MeSH