- Leave the plate without cover to air-dry the beads. Dry bead
pellets will have tiny cracks (seeNote 18). - Add 17μL EB; flush toward the beads.
- Take the well plate off the magnetic stand.
- Pipet up and down ten times and incubate for 2 min at room
temperature (cover). - Place the plate back on the magnetic stand for 2 min (cover).
- Collect a slightly smaller volume (16μL), to not transfer any
beads with the eluted DNA, and place this into low binding
tubes. - Perform quality and quantity control of cDNA libraries using a
Bioanalyzer instrument (seeFig. 4 for examples of cDNA
profiles) or measure concentration in the next step using a
Qubit instrument.
Fig. 4cDNA library profiles generated from mouse and human LCM dissected cells. (a, b) Examples of
Bioanalyzer profiles of cDNA libraries generated from mouse spinal motor neurons after HistoGene staining
from 60 and 1 cells, respectively. Libraries from 10 to 150 mouse cells typically show a peak between 800 and
1200 bp. Depending on the degradation status/quality of the tissue, the curve can be flatter or show a more
pronounced peak than in the 60 cell example depicted here. Libraries from singe cells typically have a barely
recognizable peak at around 1200 bp and the concentration can be as low as 0.1 ng/μL (100–9000 bp range).
(c, d) Human cDNA profiles typically indicate markedly more RNA degradation as reflected by a peak shift to
smaller fragment sizes or (c), a flat curve without a distinct peak (d) and in general lower cDNA yield.
FU¼fluorescence units (amount of cDNA), bp¼base pairs (fragment size)
106 Susanne Nichterwitz et al.