Profiling of gene transcripts in human platelets: An update of the platelet transcriptome

More than two years have past since publication of human platelet RNA profiles by our group and others [1–3]. Since then, significant progress has been made concerning the complexity and accuracy of microarray systems for gene expression profiling. All known, unknown and hypothetical genes identified so far in the human genome can be analysed now by the use of ‘whole genome’ microarray systems, such as the 44K Human Whole Genome Oligo Microarray (Agilent Technologies, Waldbronn, Germany). This microarray system encompasses 44,290 oligonucleotide probes spotted on a single glass slide. Excluding control and replicate probes it enables the analysis of 34,039 human gene transcripts and transcript variants. We now applied the whole genome array system to re-investigate the platelet transcriptome and provide a complete list of the RNA data. For our investigation platelet RNA was obtained from leukocyte-depleted platelet concentrates (PCs) as described in our original publication [2]. In brief, leukocyte-depleted PCs were obtained after pooling of 4 buffy coats (each derived from 500mL CPD-stabilized whole blood donations) and a total of three leukocyte depletion steps by using leukocyte reduction filters (Autostop BC, Canada; Pall, Dreieich, Germany). The microarray analysis was performed on 4 individual PC preparations representing 16 individual blood donors, all Caucasians. The 10 male and 6 female blood donors had a mean age of 38 (27 to 59) years. RNA-labelling and hybridization was done according to the protocol of the array manufacturer (Agilent Technologies). Hybridization signals were detected by using a laser scanner system (GMS418; Affymetrix Europe, High Wycombe, UK). Based on the mean fluorescence intensity (MFI) of the positive control spots in all experiments (2,995 499) we defined MFI ranges for the evaluation of the hybridization signals: positive signals – 43,500 (meanþSD); grey area – 2,500– 3,500 (mean SD); negative signals – 52,500 (mean SD). We found 7,301 (21.5%) of the 34,039 genes with hybridization signals in the positive range. Another 1,987 (5.8%) genes revealed signals in the grey area range. The MFI of the remaining 24,751 (72.7%) genes were in the negative range and indicated absence of the corresponding transcripts in platelets. A complete list of genes investigated by microarray hybridization analysis can be downloaded from the website of our institute (www.ma.uni-heidelberg.de./ inst/iti/plt-44k-profile.xls). The published list (ordered by descending MFI values) includes gene names, a short gene description with mRNA reference numbers and the MFI values (mean SD) obtained in the 4 individual microarray experiments. MFI values of positive signals are given as bold numbers, signals in the grey area range are highlighted with italicized numbers, and plain numbers indicate negative RNA signals. Genes were grouped into functional categories on the basis of the known or suspected function given in the gene list provided by the array manufacturer. The number of positive genes in several categories, such as receptors or integrins and glycoproteins was significantly higher compared to the overall number of positive genes (Table I). The over-representation