Myeloid Leukemia

226 Kohlmann et al.
3.3.11. Quality Assessment
Quality assessment is critical in obtaining reproducible microarray results.
A series of quality control (QC) procedures can be performed at various key
checkpoints during the gene expression profile analysis and include both monitoring
of sample-related parameters and technical features.
• Gel electrophoresis according to standard protocols (7) can be performed to detect
any degradation of input total RNA.
• After the IVT and cleanup of the cRNA, the ratio of 260/280 absorbance values
is assessed by spectrophotometer measurements. Good-quality cRNA should
demonstrate ratios of 1.9 to 2.1. Low cRNA yield can be a sensitive indicator of
problematic labeling procedures and/or starting material.
• Basic microarray image analyses include visual array inspections (.dat file) and
check for a correct grid alignment at each of the four corners and the center of the
array.
• Basic raw data analyses include parameters to monitor the overall background
intensity, scaling factor, percentage of present called genes (%P), and 3'/5' ratio
for the GAPD gene (see Note 8).
A technically acceptable gene expression profile can be defined according
to the following characteristics: ≥1.0 µg of input total RNA results in sufficient
cRNA yield (≥20.0 µg), concentration (≥0.6 µg/µL), and ratio of absorbance at
260 nm/280 nm (approx 2.0). The scanned array image should not show largely
visible artifacts and should have a correct grid aligned for feature extraction.
After adjusting the scanned image to common target intensity, the scaling factor
within a project should lie within two standard deviations of the mean.
When analyzing Affymetrix A-series microarrays, the %P called probe sets
should be ≥30.0%, and the 3�/5� ratio for GAPD should be ≤3.0. If the 3�/5�
ratio is >3.0, but still >30.0% of the genes were called present, the profile may
be rated as acceptable. However, in most cases with 3�/5� ratio > 3.0, the %P is
also