The average cRNA yield (Figure 1) from 1 µg of total RNA (shown in blue) was 83.1 µg. The average cRNA yield from 2 µg of total RNA (shown in red) was 135.6 µg for plate 1 and 104.4 µg for Plate 2. The normalization target was 21.8 µg, and the post normalization CVs were <10%. The error bars show the highest and lowest yields in each group.
cRNA samples were analyzed on the Agilent 2100 Bioanalyzer (Figure 2). Ambion’s RNA 6000 Ladder was used as the marker. The peak sizes are 0.2, 0.5, 1.0, 2.0, 4.0, and 6.0 kb for peaks one through six. Electropherograms showed similar nondegraded cRNA profiles with cRNA samples amplified from 1 µg and 2 µg of total RNA.
A total of 48 cRNA samples were hybridized to the Affymetrix Human Genome U133 Plus 2.0 Array. Samples were run on two different Biomek 3000 ArrayPlex instruments by two different technicians and were hybridized using multiple lots of GeneChips on different days. The data showed good correlation and reproducibility between samples.
Log correlation comparison analysis of samples with the varying amount of total RNA was prepared using GCOS 1.3 (Figure 3). The blue diagonal bars indicate a twofold change. The correlation comparison of two samples within the blue diagonal bars indicates good correlation from sample to sample, plate to plate, and sample concentration to sample concentration.
This study shows that automation can deliver consistent plate-to-plate, sample-to-sample yields. Confirming 2100 Bioanalyzer electropherograms showed similar nondegraded profiles for cRNA generated from 1 and 2 µg of total RNA, indicating no performance bias with different amounts of starting materials. The highly automated steps reduce errors, reagent waste, and deliver reproducible results.