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

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.

Reduction of synapsin in the hippocampus of patients with bipolar disorder and schizophrenia

“…21 However, the use of housekeeping genes as standards of reference for normalization, and the use of actin in particular, has recently been questioned. 36,37 Therefore, synaptophysin was employed in the present study as a reference standard and as an alternative measure of synaptic protein expression, but was not used to normalize the synapsin measurements. Actin was not measured in the present study as it was not sufficiently separated from synapsin to allow for measurement on the same blots.…”

Housekeeping genes as internal standards: use and limits

“…The synthesis of those molecules is often considered as being very few fluctuating in comparison to that of others and, by their commonplace use, are considered in many laboratories as constant and secure. But numerous studies showed that even these genes see their expression even highly does vary in given situations (Huitorel and Pantaloni, 1985;Zhang and Snyder, 1992;Goldsworthy et al, 1993;Hobbs et al, 1993;Bhatia et al, 1994;Bereta and Bereta, 1995;Lemay et al, 1996;Chang et al, 1998). This may partly be explained by the fact that housekeeping proteins are not only implicated in the basal cell metabolism but also participate in other functions (Petersen et al, 1990;Singh and Green, 1993;Ishitani et al, 1996).…”

“…The most often considered and used housekeeping genes are those for albumin (for hepatocytes) (Goldsworthy et al, 1993), b-, g-actins (Choi et al, 1991;Wei et al, 1997), cyclophilin (Bjarnason et al, 1998;Jaschke et al, 1998), G3PDH (Petersen et al, 1990;Tang et al, 1996), a-, b-tubulins (Choi et al, 1991;Serels et al, 1998), hypoxantine phosphoribosyltransferase (HRPT) (Marten et al, 1994;Foss et al, 1998), L32 for other cell types (Lemay et al, 1996;Wu et al, 1999) or 18S, 28S rRNA (Finnegan et al, 1993;Bhatia et al, 1994). The essential functions of these molecules are variable (Table 1).…”

Changes in rat liver gene expression induced by thioacetamide: Protective role ofS-adenosyl-L-methionine by a glutathione-dependent mechanism

“…Animals treated by carbon tetrachloride or undergoing partial hepatectomy show an increase in aspartate transaminase and ALT plasma activity, with a maximum at 6 hours. 27 Because SAM prevented liver gene expression changes induced by TAM, this effect being blocked by BSO, we decided to see if similar results assaying the ALT serum activity, a parameter of liver injury, were obtained. TAM-treated animals had increases in the serum ALT activity with a peak around FIG.…”