Myeloid Leukemia

220 Kohlmann et al.
adding 175 µL ammonium acetate (7.5 M), 0.5 µL glycogen (20 mg/mL), and
1000 µL of absolute ethanol (see Note 4). Store overnight or longer at –20°C.
5. Pellet the ds cDNA by centrifugation at maximum speed for 30 min, discard the
supernatant carefully. Wash the pellet by overlaying with 500 µL 80% ethanol.
Centrifuge at maximum speed for 15 min. Then discard the supernatant carefully.
6. Wash the ds cDNA pellet by overlaying with 500 µL 80% ethanol. Centrifuge at
maximum speed for 15 min. Then discard the supernatant carefully.
7. Wash the ds cDNA pellet by overlaying with 500 µL 100% ethanol. Centrifuge at
maximum speed for 15 min. Then discard the supernatant carefully.
8. Air dry the pellet by evaporating residual ethanol. This takes approx 5–10 min.
9. Dissolve the cDNA pellet in 22 µL nuclease-free water and vortex for 10 s. Continue
immediately with the IVT procedure.
3.3.4. Synthesis of Biotin-Labeled cRNA
After the ds cDNA has been purified, it is transcribed in vitro to generate
more than 400 biotinylated cRNA molecules for each ds cDNA molecule. Adequately
intact input RNA should result in an expected yield of biotinylated
cRNA of between 4- and 10-fold greater than the total RNA input (3).
1. Pipet the template cDNA and reaction components from the RNA transcript
labeling kit into RNase-free microcentrifuge tubes (see Table 4 and Note 5).
Perform all steps at room temperature to avoid precipitation of DTT.
2. Carefully mix the reagents and collect the mixture in the bottom of the tube by
brief centrifugation (5 s). Then place the reaction tube in a 37°C incubator and
incubate for 5 h. After the IVT, immediately proceed with the purification of the
biotin-labeled cRNA.
3.3.5. Cleanup of Biotin-Labeled cRNA
After the IVT reaction, cleanup of biotinylated cRNA is performed according
to the RNeasy Mini Kit protocol (Qiagen). GITC-containing lysis buffer
and ethanol are added to the sample to create conditions that promote selective
binding of the cRNA to the silica-gel membrane in the RNeasy mini column.
The cRNA binds to the membrane, contaminants are efficiently washed away,
and high-quality cRNA is eluted in water. Eight individual samples can be
processed in parallel. All steps of the RNeasy protocol should be performed
quickly at room temperature. All centrifugation steps can be performed in a
standard microcentrifuge. RPE wash buffer is supplied as a concentrate. Before
using it for the first time, 4 vol of absolute ethanol are added to obtain a
working solution. According to the manufacturer’s recommendation, RLT
buffer is prepared freshly for each cleanup procedure (10 µL 2-mercaptoethanol
per 1 mL RLT buffer; mixed in a 15-mL Falcon tube).
1. Adjust the sample to a volume of 100 µL with water. Therefore, add 60 µL water
to the 40-µL cRNA reaction volume.