The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<10 US dollars) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a beta-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a beta-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of beta-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r(2) = 0.6802) between the array and the RT-PCR beta-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean +/- SD, 0.34 +/- 0.09) and degraded (1.71 +/- 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.