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Blocking of Globin Reverse Transcription to Enhance Human Whole Blood Gene Expression Profiling
Gene expression profiling of human blood is becoming increasingly used in the pharmaceutical and diagnostic industries for the discovery and development of clinical biomarkers linked to human disease. The laboratory steps and equipment required to reproducibly isolate White Blood Cells (WBCs) not only increase the cost but also the elapsed time to RNA isolation. The manipulations required and time to RNA isolation likely result in altered gene expression profiles. To mitigate this issue, methods have been developed to process whole blood prior to RNA isolation and gene expression analysis. The most useful microarray expression profiling data is generated from whole blood samples in which Red Blood Cells (RBCs) are selectively pre-lysed and the RNA is subsequently purified. However, the abundance of globin mRNA transcripts in RBCs and reticulocytes masks the mRNA contributed by WBCs.
Gene Logic's globin reduction method utilizes 2’O-methyl modified oligomers as gene-specific blockers that greatly reduce the ability of reverse transcriptase to generate globin cDNAs during the sample preparation process. The blocking procedure utilizes a proprietary mixture of oligomers that have been optimized for use with 2-5 ìg of total RNA from whole blood.
In this report, we compare microarray data from WBCs (the gold standard in terms of low globin mRNA contamination), unblocked whole blood and blood samples in which globin cDNA synthesis is blocked using Gene Logic’s globin reduction protocol.
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