Detection of gene fusions in acute leukemia using bead microarrays

This unit describes a method for the rapid and simultaneous detection of hybrid mRNAs (hRNA; also known as gene fusions, RNA fusions, or chimeric RNA), resulting from recurrent chromosome translocations or inversions in acute leukemia. The principal components of the assay are a multiplex RT-PCR, which simultaneously amplifies any of a number of possible hRNA targets, and a liquid bead microarray (LBMA), which is capable of unambiguously identifying the amplified hRNA. The entire procedure from RNA extraction to display of results can be broken into four steps: (1) conjugated-microsphere and LBMA manufacture and quality control, (2) cDNA synthesis, (3) multiplex PCR, and (4) hybridization of multiplex PCR products to the hRNA-detecting LBMA and assay read-out on a flow cytometer. The assay is designed to detect the most common risk-stratifying translocation in pediatric acute lymphoblastic leukemia, satisfying the demand for inclusion of genetic data in the diagnosis of acute leukemia as predicated by the current WHO classification of hematopoietic and lymphoid neoplasms.