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E. RNA SOURCE, RNA QUALITY AND ARRAY REPRODUCIBILITY
| We assessed total RNA
integrity by electrophoresis using the Agilent Bioanalyzer
(Agilent, Palo Alto, California). Additional inclusion
criteria for accepting expression array results that
were at least 10% of genes on an array were expressed
(“present call” by MAS 5.0), GAPDH and Actin
3’/5’ ratio less than 5, and a scaling factor
within four standard deviations of the mean of all 120
chips analyzed in this study. We isolated mononuclear
(leukemia) cells from each bone marrow aspirate by centrifugation
over Ficoll. The median percent blasts (post-Ficoll)
was 97% in the diagnostic bone marrow samples and 95%
in post-treatment samples. All post-treatment samples,
except four, had greater than 65% blasts. The four with
less than 65% blasts (i.e. 41%, 49%, 60%, 62%) had 78%,
94%, 94% and 98% blasts in their diagnostic samples.
The percent blast was not determined in three post-treatment
samples, but all had greater than 98% blasts at diagnosis.
While differences in post-treatment expression could
be influenced by selection of cells that favored survival
after treatment, the delayed cytotoxicity of these antimetabolites,
coupled with the modest drop in circulating blast counts
at the time the post-treatment samples were obtained
(median 9.7% decrease across all samples), indicate
that selection could not explain the magnitude of changes
in gene expression that we observed. Furthermore, the
percentage of ALL blasts in the pre- and post-treatment
samples did not differ (median of 97% at diagnosis versus
95% post-treatment).
We extensively assessed reproducibility of gene expression
within our facility, as previously described (Cancer
Cell 1, 133-143 (2002)). Additionally, we tested duplicate
cryopreserved bone marrow samples from two patients
obtained after treatment; RNA was extracted approximately
six months apart. Replicate analysis of the same sample
produced similar results, as depicted in Fig. 7 (i.e.,
R2=0.87 and 0.92).
|
Fig.
7: Concordance between replicate gene expression experiments.
Expression level of two separate aliquots of post-treatment bone
marrow, obtained from two different patients, were extracted and
analyzed independently, revealing a high level of concordance between
replicate analyses.
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