Myeloid Leukemia

Classification of AML by DNA-Oligonucleotide Microarrays 221
Table 4
In Vitro Transcription Reaction
Component Volume
Reaction buffer, 10X concentrated 4 µL
Dithiothreitol, 10X concentrated 4 µL
RNase inhibitor mix, 10X concentrated 4 µL
Biotin-labeled ribonucleotides, 10X concentrated 4 µL
T7 RNA polymerase, 20X concentrated 2 µL
Template ds cDNA Variable
Water Variable (to give a final volume
of 40 µL)
Final volume 40 µL
2. Add 350 µL RLT buffer and mix thoroughly. The total volume now is 450 µL.
3. Add 250 µL absolute ethanol to the diluted cRNA, and mix thoroughly by
pipetting. Do not centrifuge. The total volume now is 700 µL.
4. Continue immediately to apply the sample to an RNeasy mini column placed in a
2-mL collection tube. Close the tube gently, and centrifuge for 15 s at 8000g.
5. Apply the flow-through again to the same column placed in a new 2-mL collection
tube. Close the tube gently, and centrifuge for 15 s at 8000g. Now discard
the flow-through. Transfer the RNeasy column into a new 2-mL collection tube.
6. Pipet 500 µL RPE buffer onto the column. Close the tube gently, and centrifuge
for 15 s at 8000g to wash the column. Discard the flow-through. Transfer the
column into a new 2-mL collection tube.
7. Add another 500 µL RPE buffer to the column. Close the tube gently, and centrifuge
for 2 min at ≥8000g to dry the membrane. Place the column in a new 2-mL
collection tube, and discard the old collection tube with the flow-through. Centrifuge
in a microcentrifuge at full speed for 1 min.
8. To elute, transfer the column to a new 1.5-mL collection tube (Eppendorf). Pipet
40 µL nuclease-free water directly onto the membrane and incubate for 1 min.
Close the tube gently, and centrifuge for 1 min at 8000g to elute.
Store the isolated cRNA on ice while aliquots are pipetted for downstream
applications. The concentration of cRNA is determined by measuring the absorbance
at 260 nm (A 260) in a spectrophotometer. In general, to ensure significance,
readings should be between 0.10 and 1.0. An absorbance of 1 U at 260
nm corresponds to 40 µg of cRNA per mL. The isolated cRNA is diluted 1:50
for the measurement in nuclease-free water (2 µL total RNA, 98 µL water).
The A 260/A 280 ratio should be close to 2.0 (ratios between 1.9 and 2.1 are acceptable).