Glass needle-based chromosome microdissection (midi) was first described in the 1980s [1]. It is a straightforward approach for setting up species specific whole and partial chromosome painting probes suited for fluorescence in situ hybridization (FISH) [2],[3]. Furthermore, DNA-isolation by midi can be the starting point for many other studies, including exact breakpoint characterization by next generation sequencing [4],[5],[6].

Compared to chromosome flow sorting [7], midi needs less sophisticated and expensive equipment and is not only restricted to production of whole chromosome paints. Concerning laser-based microdissection compared to midi the former is not regularly applied yet for preparation of whole and not at all for partial chromosome painting probes [8]. Furthermore, just recently we published a protocol how to use semi-archived material in midi [8].

Accordingly, midi is a very timely approach. Even though we provided earlier detailed protocols on how to do midi [3], practical guidance on how to do hands on steps of this procedure in more detail still lack. Thus, in this article, supplied with the accompanying movie, we are sure to be able to close this gap and to give advice on how to do midi under real conditions.

THE MIDI PROCEDURE AS SHOWN IN THE ACCOMPANYING MOVIE

The midi procedure as shown here is based on the protocol published in ref. [3]. Special emphasis is given in this paper on some working steps of midi procedure, which were not outlined in detail there.

HOW TO DO GLASS NEEDLE AND PIPETTE PREPARATION?

The preparation of glass needles and pipettes must be done by using a pipette puller. This machine is shown in Figure 1 and works with a heating unit and weights and balances. Glass rods or pipettes can be fixed in there and pulled in length as shown in Video 1.

Also the necessary silanization procedure of extended pipettes (Figure 2) is explained and shown in Video 1. This step is essential to avoid sticking of the microdissected DNA to pipettes, instead of being regularly processed during the procedure.

HOW TO PREPARE COVERSLIPS AS CARRIERS OF CHROMOSOMES?

Due to optical features of applied inverted microscopes one cannot use microscope slides for midi. Instead, only sodium dodecyl sulfate (SDS)-treated coverslips (Figure 3) can be applied to spread and fix chromosomes on them. The spreading of the chromosomes on the coverslips can be improved by holding them in the steam of a water bath. Very important is to mark the orientation “up-side” and “down-side” of the coverslip, which can be done by breaking off an edge (Figure 4). Before optical control of metaphase spreading, coverslip goes for 4 minutes on a heating plate (60 °C).

HOW TO DO CHROMOSOME G-BANDING ON THE COVERSLIP?

G-banding, if necessary, is done according to standard protocols [9]. Main difference to regular protocols is that coverslip staining is done here in 50 mL tubes (Figure 5).

HOW TO PREPARE THE INVERTED MICROSCOPE?

The inverted microscope used, being equipped with one, or better two micromanipulating units, has to be prepared by mounting elongated pipette and glass needle at the corresponding spots. The pipette has to be loaded with the collection drop [3] before mounting (Figure 6 and Figure 7).

HOW TO DO MIDI?

The exact use of the micromanipulators is a matter of personal training. However, the principle is simple: just cut out a chromosomal band/region or scrape together one whole chromosome to a single spot. Afterward pick up the microdissected DNA by the glass needle. Then the DNA sticking to the glass needle is transferred to the collection drop in the pipette and washed off.

DNA AMPLIFICATION

The DNA accumulated in the collection drop after 1–15 midi steps is then transferred into a reaction tube. This is subjected to DNA probe amplification by a specific PCR procedure [degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)] [3].

Midi is a laborious, manual, and sophisticated approach, needing some preparation to be performed. However, it is still the best suited, most straightforward, and most cost-efficient way to establish whole or partial chromosome painting probes for molecular cytogenetic studies or other studies including sequencing.