Total platelet RNA was extracted using TRIzol reagent (Invitrogen, San Diego, CA).
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's suggestion. Gene expression was performed using Q-RT-PCR with TaqMan Master Mix and TaqMan Assay Reagent (Life Technologies) [19 (link)]. Changes in MRP4, CXCL8 and ACTIN-mRNA amounts were quantified by ΔΔCt method for relative quantization of gene expression using SDS software version 2.3 (Applied Biosystems). All samples were free of mature transcript for CXCL8 (Interleukin-8), a leukocyte-specific gene product, to support that absence of leukocyte contamination [12 (link)].
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's suggestion. Gene expression was performed using Q-RT-PCR with TaqMan Master Mix and TaqMan Assay Reagent (Life Technologies) [19 (link)]. Changes in MRP4, CXCL8 and ACTIN-mRNA amounts were quantified by ΔΔCt method for relative quantization of gene expression using SDS software version 2.3 (Applied Biosystems). All samples were free of mature transcript for CXCL8 (Interleukin-8), a leukocyte-specific gene product, to support that absence of leukocyte contamination [12 (link)].
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