S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eucaryotic cells and tissues.

In the course of studies looking for differentially expressed genes, we isolated as a control a cDNA hybridizing to a highly and constantly expressed RNA. Further characterization of the hamster and human full-length cDNAs showed that they correspond to ribosomal protein S26 RNA. Sequence comparison revealed a very high degree of conservation at the proteic level amongst distantly related eukaryotic organisms such as man and Drosophila. The S26 gene is expressed as a 600-700 bases long RNA at high and constant level in human adult tissues and in mammalian cell lines cultured in different physiological contexts. This mRNA therefore represents a very suitable invariant standard for all experiments involving RNA quantification. Only a few probes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), /3-actin and /32-microglobulin, are available for controlling RNA levels on Northern blots, in RNAse mapping procedures or run-on assays. Several years ago, we isolated and used a set of GAPDH cDNAs as a control to monitor gene regulation experiments, as the level of the corresponding RNA remains unaltered in cells treated by various drugs, such as cycloheximide, or actinomycinD (1 —3). Since then, the use of GAPDH probe became so widespread that detection kits have been made available for the monitoring of RT-PCR reactions. Nevertheless, in some instances, the use of this probe suffers several drawbacks: i) GAPDH RNA accumulates in cells exposed for several hours to various hormones or growth factors such as Insulin (4), Epidermal Growth Factor or Fetal Calf Serum (5), ii) there is some degree of tissue specific expression of total and poly (A) GAPDH RNA in rat and in man (6), and iii) its size (1,3 kb) is within the average of mRNA size distribution, and might impair a proper detection of low abundance class mRNA during subsequent hybridization of Northern membranes.