Chromosome Microdissection on Semi-Archived Material

Abstract

Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Keywords: DNA; air-drying method; chromosome; glass-needle-based chromosome microdissection (midi); molecular cytogenetics; semi-archived material.