The influence of RNA quality categorization obtained with both user-independent classifiers on gene expression profiling was explored using real-time RT-PCR. The expression levels of three housekeeping genes (HKG)-GAPDH, GUSB and TFRC-were measured in 16 aliquots of a unique RNA displaying various integrity metrics ( Table 1 ). The mean correlation coefficient (r) between the threshold cycle (Ct) among the 16 samples and both quality metrics was found high: r = À0.87 considering the RIN metrics and r = 0.85 considering the DegFact values. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ). Considering that HKG expression was measured relative to the reference sample, an obvious decline of the relative expression levels was observed, up to 24, 70 and 82%, in samples categorized according to the RIN metrics ( Figure 5A) and DegFact values ( Figure 5B ). These results indicate that 2-to 7-fold differences may be expected in the relative expression levels of genes in samples that differ only by their quality (Table 2 ). These fold differences are much larger than those measured for RNA samples of comparable integrity, consistently lower than 1.6 (Table 2 and Figure 5 ). In addition, an unambiguous gap in the distribution may be defined ( Figure 5A and B) , distinguishing the RNA samples of the higher quality categories (RIN > 8 and DegFact values < 7) from those of the lower categories (RIN < 8 and DegFact values > 12).