Methamphetamine alters reference gene expression in nigra and striatum of adult rat brain

Abstract

The nigrostriatal dopaminergic system is a major lesion target for methamphetamine (MA), one of the most addictive and neurotoxic drugs of abuse. High doses of MA alter the expression of a large number of genes. Reference genes (RGs) are considered relatively stable and are often used as standards for quantitative real-time PCR (qRT-PCR) reactions. The purpose of this study was to determine whether MA altered the expression of RGs and to identify the appropriate RGs for gene expression studies in animals receiving MA. Adult male Sprague-Dawley rats were treated with high doses of MA or saline. Striatum and substantia nigra were harvested at 2h or 24h after MA administration. The expression and stability of 10 commonly used RGs were examined using qRT-PCR and then evaluated by geNorm and Normfinder. We found that MA altered the expression of selected RGs. These candidate RGs presented differential stability in the striatum and in substantia nigra at both 2h and 24h after MA injection. Selection of an unstable RG as a standard altered the significance of tyrosine hydroxylase (TH) mRNA expression after MA administration. In conclusion, our data show that MA site- and time-dependently altered the expression of RGs in nigrostriatal dopaminergic system. These temporal and spatial factors should be considered when selecting appropriate RGs for interpreting the expression of target genes in animals receiving MA.

Keywords: Housekeeping genes; Methamphetamine; Nigra; Real-time PCR; Reference genes; Striatum.

Fig.1. geNorm analysis of reference genes in striatum tissue at (A) 2h and (B) 24h after injection of MA or saline

Average expression stability values (M) was used to identify stable RGs. Lower M values (Right) indicate a more stable expression.

Fig.2. geNorm analysis of reference genes in nigra tissue at (A) 2h and (B) 24h after last dose of MA or saline

Average expression stability values (M) start from the least stable gene at left and end with most stable genes at the right. Lower M values indicate more stable expression.

Fig.3. Stability values of candidate reference genes ranked by Normfinder

The gene with the lowest stability value has the most stable expression; the gene with the highest value the least stable expression.

Fig 4. Relative TH mRNA levels measured by normalization to different reference genes

The comprehensive ranking of the most to the least stable genes recommended by RefFinder are respectively from left to right in the X-axis. The expression levels of the saline group are set to 1. (*p<0.05, Student's t-test, n=8 for each group)

Fig.5. Relative mRNA level of Hmbs and Ubc (normalized to Rps6 and B2m) in Striatum at 2h after last injection of MA and saline

The expression levels of saline group are set to 1 with all the data normalized to Rps6 and B2m, which are used as the best reference gene pair. (A) Comparison of relative Hmbs mRNA levels between saline and MA groups. (B) Comparison of relative Ubc mRNA levels between saline and MA groups. (* p<0.01, Student's t-test, n=8, each group)