Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation

Authors


Pathology Associates Inc., 4915 D Prospectus Dr., Durham, NC 27713, USA.

Abstract

Quantitative knowledge of gene expression can provide valuable information for understanding the action of chemicals that alter cell proliferation and cancer. Accurate quantification of mRNA levels requires the normalization of the gene of interest to a gene with transcriptional levels that do not vary through the cell cycle or with a particular treatment. Changes in expression were examined in proliferating or non-proliferating rat liver for three constitutively expressed ‘housekeeping’ genes commonly used to normalize mRNA levels from Northern blots. In addition, a direct method of quantifying poly(A)+ mRNA by hybridization with a radiolabelled polythymidylate—poly(T)—probe was compared with traditional methods. Hepatocyte cytolethality and a subsequent wave of hepatocyte proliferation were induced in male Fischer-344 rats by treatment with a single gavage dose of carbon tetrachloride. Induced cell proliferation peaked at 48 h after treatment. Expression of the housekeeping genes actin, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and albumin, as well as the proto-oncogene H-ras, was determined by Northern blot analysis at times from 0.5 h to 4 days after treatment. Time-dependent changes were observed in the expression of these genes relative to the levels observed in the untreated control animals. Actin expression peaked at 3.4-fold over control and GAPDH expression was increased by 1.9-fold over control. Albumin mRNA levels varied the least, 1.4-fold over control, indicating that this gene is more appropriate than actin or GAPDH for normalization of proto-oncogene expression under experimental conditions that induce cell proliferation in rat liver. The direct quantification of poly(A)+ mRNA using a poly(T) probe was not influenced by the induction of cell proliferation. This method may be useful when the expression of housekeeping genes is affected by treatment.

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