Abstract
BackgroundAlternative mRNA processing mechanisms lead to multiple transcripts (i.e. splice isoforms) of a given gene which may have distinct biological functions. Microarrays like Affymetrix GeneChips measure mRNA expression of genes using sets of nucleotide probes. Until recently probe sets were not designed for transcript specificity. Nevertheless, the re-analysis of established microarray data using newly defined transcript-specific probe sets may provide information about expression levels of specific transcripts.Methodology/Principal FindingsIn the present study alignment of probe sequences of the Affymetrix microarray HG-U133A with Ensembl transcript sequences was performed to define transcript-specific probe sets. Out of a total of 247,965 perfect match probes, 95,008 were designated “transcript-specific”, i.e. showing complete sequence alignment, no cross-hybridization, and transcript-, not only gene-specificity. These probes were grouped into 7,941 transcript-specific probe sets and 15,619 gene-specific probe sets, respectively. The former were used to differentiate 445 alternative transcripts of 215 genes. For selected transcripts, predicted by this analysis to be differentially expressed in the human kidney, confirmatory real-time RT-PCR experiments were performed. First, the expression of two specific transcripts of the genes PPM1A (PP2CA_HUMAN and P35813) and PLG (PLMN_HUMAN and Q5TEH5) in human kidneys was determined by the transcript-specific array analysis and confirmed by real-time RT-PCR. Secondly, disease-specific differential expression of single transcripts of PLG and ABCA1 (ABCA1_HUMAN and Q5VYS0_HUMAN) was computed from the available array data sets and confirmed by transcript-specific real-time RT-PCR.ConclusionsTranscript-specific analysis of microarray experiments can be employed to study gene-regulation on the transcript level using conventional microarray data. In this study, predictions based on sufficient probe set size and fold-change are confirmed by independent means.
Highlights
DNA microarrays are important experimental tools to gain knowledge about the steady state levels of mRNA species
Predictions based on sufficient probe set size and foldchange are confirmed by independent means
Affymetrix GeneChips were designed to contain a series of oligonucleotide probes complementary to a specific mRNA of known genes
Summary
DNA microarrays are important experimental tools to gain knowledge about the steady state levels of mRNA species. Affymetrix GeneChips were designed to contain a series of oligonucleotide probes complementary to a specific mRNA of known genes. MRNA processing mechanisms can lead to different transcripts of the same gene which can have specific biological functions [1,2]. Methods that apply microarray profiling would – by using the available and measured transcript-specific probes – provide additional information not on the expression levels of a gene and the respective splice isoforms. Alternative mRNA processing mechanisms lead to multiple transcripts (i.e. splice isoforms) of a given gene which may have distinct biological functions. Microarrays like Affymetrix GeneChips measure mRNA expression of genes using sets of nucleotide probes. The reanalysis of established microarray data using newly defined transcript-specific probe sets may provide information about expression levels of specific transcripts
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