Microarray analysis of human tissue is frequently hindered by the limited amount of
RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total
RNA derived from cultured renal epithelial cells from
tuberous sclerosis complex (
TSC) carriers and control individuals was amplified by in vitro transcription with
T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified
RNA (aRNA)) were analyzed as direct replicates and
dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total
RNA), 0.85 (40 microg total
RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total
RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total
RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total
RNA is proportional to the amount of
RNA used and underscore the requirement of large amounts of total
RNA for a comprehensive characterization of gene expression profiles.
RNA amplification allows the detection of a large number of genes expressed in the starting
RNA population without altering their relative intensities significantly. Thus, an
RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of
RNA, including those extracted from limiting clinical samples and microdissected histological specimens.