Paxgene blood rna kit 50

Blood samples were collected in RNA-stabilizing PAXgene tubes (PreAnalytix, BD, Heidelberg, Germany), and stored at −80 °C until analysis. RNA was isolated with the PAXgene Blood RNA Kit 50 (PreAnalytix, Qiagen, Hilden, Germany) according to standard protocols. Total RNA yield was determined using the Quant-iT RiboGreen RNA Reagent and Kit (Life Technologies, Darmstadt, Germany), and a Wallac Victor 2 1420 Multilabel Counter (Perkin Elmer, Rodgau, Germany). Total RNA purity was evaluated via OD measurements (260 nm/280 nm) in a NanoDrop (peqLab, Erlangen, Germany). RNA integrity was determined by RNA integrity number measurement using RNA 6000 Nano Assay RNA chips run in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The inclusion criteria were as follows: a ratio of 1.9–2.2 (OD 260/280); RNA integrity number>8.0, and the absence of a peak of genomic DNA contamination in electropherograms. Samples were transcribed to cDNA and hybridized to an Affymetrix Gene Chip Human Exon 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) using the Whole Transcript Sense Target Labeling Assay protocol and 100 ng of total RNA from each sample.