Computational protocol: Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia

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Protocol publication

[…] To access the expression of R2 splice variant we analyzed ten transcriptomes of cytogenetically normal AML and FACS-sorted bone marrow samples of three healthy individuals. Bone marrow of healthy donors was sorted by FACS as previously described []. Diagnostic AML samples were collected from 10 adult patients with cytogenetically normal karyotype enrolled on German-Austrian AML Study Group (AMLSG) treatment protocols for younger adults [AMLSG 07-04 (NCT00151242)]. Written informed consent was obtained from all patients and healthy donors, and the gene expression study was approved by the IRB.Total RNA was isolated using AllPrep DNA/RNA Kit (Qiagen). RNA integrity was assessed on Agilent Bioanalyzer using Agilent RNA 6000 Pico or Agilent RNA 6000 Nano Kit (Agilent Technologies) and samples with RNA integrity number (RIN) of at least 7.5 were selected for RNA-seq. A total of 1 μg of RNA was rRNA depleted and sequencing libraries were obtained using Ribo-Zero Gold rRNA Removal Kit (human; Illumina) according the manufacturer protocol. The transcriptomes were sequenced on HiSeq2500 (Illumina) and on average 63.2 million reads were obtained per sample. The reads were aligned to reference genome ucsc.hg19 using STAR aligner []. Reads mapping to the unique exon of R2 NPM1 with the coordinates chr5:170833400-170833731 of hg19 were quantified using bedtools with options intersect and split. The counts were RPKM normalized, log2 transformed and plotted in GraphPadPrism software. […]

Pipeline specifications

Software tools STAR, BEDTools, GraphPad Prism
Applications Miscellaneous, RNA-seq analysis
Organisms Homo sapiens
Diseases Leukemia, Myeloid, Acute