Illumina HiSeq 2000 sequencing; Molecular characterization of acute lymphoblastic leukemia with CRLF2 overexpression in childhood
Molecular characterization of acute lymphoblastic leukemia with CRLF2 overexpression in childhood
In accordance with institutional review board regulations, clinical samples were obtained from 1-18 years old children and adolescents with ALL before treatment. Diagnostics, risk group assignment, and treatment were performed according to the AIEOP-BFM ALL 2000 protocol (Conter, et al. 2010; Flohr, et al. 2008) and subsequently according to the AIEOP-BFM ALL 2009 protocol (EudraCT Number: 2007-004270-43). The study was approved by the institutional review board of the Hannover Medical School, Hannover, Germany and the University Medical Center Schleswig-Holstein, Kiel, Germany, and informed consent was obtained from patients and/or their legal guardians in accordance with the Declaration of Helsinki. Total RNA (including small RNA) was extracted from both PAXgene blood tubes using the PAXgene blood miRNA extraction kit (Qiagen) according to the manufacturer’s protocol. The RNA concentration and quality in each sample was evaluated by the RNA HS Assay Kit using the Qubit Fluorometer (Life Technologies) and the RNA Nano Chip using the 2100 Bioanalyser (Agilent), respectively. Total RNA preparations with an RNA integrity number (RIN) number >7.5 and sufficient quantity were used for the mRNA expression analysis. Globin depletion was performed using the GLOBINclear kit (Ambion). MACE and STDGE libraries were prepared as previously described using the MACE kit by GenXPro GmbH (Germany). Briefly, polyadenylated RNA was extracted from 2 μg Globin-depleted total RNA and reverse-transcribed with biotinylated poly(dT) primers. The resulting cDNA was fragmented and biotinylated ends were captured by streptavidin beads. TrueQuant DNA adapters (GenXPro) were ligated. to the enriched ends, and subsequent to PCR amplification the libraries were sequenced on the HiSeq2000 platform (Illumina).
Experimental Factor: P2RY8-CRLF2 fusion negative
genotype