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Classification of AML by DNA-Oligonucleotide Microarrays 217<br />
sample, including any precipitate that may have formed, to an RNeasy mini column<br />
placed in a 2-mL collection tube. Close the tube gently and centrifuge for 15<br />
s at 8000g. Discard the flow-through. Transfer the column into a new 2-mL collection<br />
tube.<br />
4. Add 700 µL washing buffer RW1 to the column. Close the tube gently, and centrifuge<br />
for 15 s at 8000g. Discard the flow-through and collection tube. Transfer<br />
the column into a new 2-mL collection tube.<br />
5. Pipet 500 µL RPE washing buffer onto the column. Close the tube gently, and<br />
centrifuge for 15 s at ≥8000g. Discard the flow-through. Transfer the column into<br />
a new 2-mL collection tube.<br />
6. Add another 500 µL RPE washing buffer to the column. Close the tube gently,<br />
and centrifuge for 2 min at ≥8000g to dry the membrane. Subsequently, to eliminate<br />
any chance of possible RPE washing buffer carryover, place the column in a<br />
new 2-mL collection tube, and discard the old collection tube with the flowthrough.<br />
Centrifuge in a microcentrifuge at full speed for 1 min.<br />
7. Remove the column from the collection tube carefully so the column does not<br />
contact the flow-through, as this will result in carryover of ethanol (see Note 3).<br />
Transfer the column to a new 1.5-mL collection tube and proceed with elution of<br />
total RNA.<br />
8. Pipet 40 µL nuclease-free water directly onto the membrane. Close the tube gently,<br />
incubate for 1 min and centrifuge for 1 min at 8000g to elute.<br />
Store the isolated total RNA on ice while aliquots are pipetted for quantification<br />
and during the subsequent cDNA synthesis step. The concentration of<br />
RNA is determined by measuring the absorbance at 260 nm (A 260) in a spectrophotometer.<br />
In general, to ensure significance, readings should be between 0.10<br />
and 1.0. An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per<br />
mL. The isolated total RNA is diluted 1:50 for the measurement in nucleasefree<br />
water (2 µL total RNA, 98 µL water).<br />
3.3.2. Synthesis of ds cDNA<br />
For the synthesis of ds cDNA, the one-tube double-stranded cDNA Synthesis<br />
System (Roche Applied Science, Mannheim, Germany) can be used. This<br />
system has been designed according to the method of Gubler and Hoffmann<br />
(2) and is optimized to reduce manipulation steps, allowing the rapid and reliable<br />
synthesis of full-length cDNAs, especially from total RNA. During the<br />
first-strand reaction, avian myeloblastosis virus (AMV) reverse transcriptase<br />
is used. The initiation of the first-strand synthesis depends upon hybridization<br />
of an oligo [(dT)24 T7promoter]65 primer to the mRNA, usually at the poly(A)<br />
tail. This primer also contains a promoter for the T7 RNA polymerase, which<br />
enables a subsequent in vitro transcription (IVT) reaction. The first- and second-strand<br />
syntheses are performed in the same tube, which speeds the synthe-
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