You are here:
VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR
Citation:
THAI, S. Y., S. D. HESTER, AND J. A. ROSS. VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR. Presented at Quantitative PCR, Microarrays, and Biological Validation, Providence, RI, October 22 - 24, 2006.
Description:
Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and relative quantification methods to identify the gene expression level changes in the lungs of mice treated with benzo[a]pyrene,
Strain A/J mice were treated with different doses (50 mg/kg, 5 mg/kg, 0.5 mg/kg and 0.05 mg/kg) of benzo[a]pyrene by i.p. injectons at 5 animals for each dose. Benzo[a]pyrene doses were dissolved in tricaprylin while control animals received tricaprylin alone. Animals were sacrificed 24 hours and 72 hours after injections. Lungs were removed, flash frozen in liquid nitrogen, and stored at -80°C until RNA extraction. RNA from controls and 50 mg/kg, 3 day treatment were used for microarray analysis using Affymetrix 430_2 mouse whole genome chips. Data were analyzed using RMA in Bioconductor R with p value set at 0.05. A subset of significantly altered genes was selected to be further analyzed by qRT-PCR in lower dose-treated mouse lungs. These genes are cyp 1a1 (Cytochrome P450 1a1), cyp 1b1 (Cytochrome P450 1b1), AHRR (aryl hyrdrocarbon receptor repressor), Ccbp2 (chemokine binding protein 2), Kit oncogene, IL-4 receptor, CCAATT/enhancer binding protein beta, Btebl (Basic transcription element binding protein 1) and Stmn2 (Stahtmin-like 2).
Using qRT-PCR, the gene expression levels (compared to non-treated controls) for cyp 1a1 after 1 day treatment were 0.8-, 5-, and 90-fold for 0.5, 5 and 50 mg/kg doses, respectively. The expression levels for 3 day treatment were 2-, 0.4- and 31-fold for 0.5, 5 and 50 mg/kg doses, respectively. When we examined the expression of the endogenous control gene beta-actin, we noticed a two-fold variation at the 5 mg/kg, 3 day treatment relative to controls. The beta-actin gene was sometimes induced 2-fold, and sometimes suppressed 2-fold at 5 mg/kg, 3 day treatment. The variation for beta-actin gene expression is greatest at 5 mg/kg for 3 day treatment. Therefore, the suppression we detected by qRT-PCR at this dose/time may or may not be correct. We have started assessing the variability in expression of various candidate endogenous control genes. From the preliminary data, it appears that 18S rRNA has relatively little variation at the 5 mg/kg, 3 day treatment. We are evaluating additional housekeeping genes to determine their utility as endogenous controls. Experiments are ongoing to determine the optimum approach to normalize the expression values of our genes of interest to accurately measure changes in their expression.