SUPPLEMENTARY DATA to: Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA (original) (raw)

Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

PLoS ONE, 2013

Background: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts.

Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction

Journal of Clinical Investigation, 1988

Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.

Platelets Purification Is a Crucial Step for Transcriptomic Analysis

International Journal of Molecular Sciences, 2022

Platelets are small anucleate cells derived from the fragmentation of megakaryocytes and are involved in different biological processes especially hemostasis, thrombosis, and immune response. Despite their lack of nucleus, platelets contain a reservoir of megakaryocyte-derived RNAs and all the machinery useful for mRNA translation. Interestingly, platelet transcriptome was analyzed in health and diseases and led to the identification of disease-specific molecular signatures. Platelet contamination by leukocytes and erythrocytes during platelet purification is a major problem in transcriptomic analysis and the presence of few contaminants in platelet preparation could strongly alter transcriptome results. Since contaminant impacts on platelet transcriptome remains theoretical, we aimed to determine whether low leukocyte and erythrocyte contamination could cause great or only minor changes in platelet transcriptome. Using microarray technique, we compared the transcriptome of platelet...

Comparative RNA expression analyses from small‐scale, single‐donor platelet samples

Journal of Thrombosis and Haemostasis, 2006

Background: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. Objectives: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. Methods: Differential hybridization to cDNA macro-arrays and suppressivesubtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix Gene-Chip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. Results: Macroarrays identified 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. Conclusions: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.

Comparative RNA expression analyses from small-scale, single-donor platelet samples1

Journal of Thrombosis and Haemostasis, 2006

Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. Differential hybridization to cDNA macro-arrays and suppressive-subtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. Macroarrays identified approximately 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.