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QUANTITATIVE PCR VALIDATION OF COPPER-REGULATED GENE EXPRESSION IN SACCHAROMYCES CEREVISIAE

Abstract

Cells must tightly regulate copper levels because it serves as an essential micronutrient and is toxic when present in excess. Abnormal copper levels are a factor in several human disorders such as Menkes disease and Wilson’s disease. Due to the similarity of pathways between yeast and other eukaryotes, budding yeast is a useful model for studying eukaryotic cell functions. Yeast cells regulate copper levels by differential expression of genes related to uptake (e.g., CTR1) and sequestration (CUP1). Previously, RNA sequence analysis (RNA-Seq) provided a list of genes that are significantly regulated by copper. RNA-Seq uses next-generation sequencing to quantify expression levels across the entire genome. However, RNA-Seq data must be verified and validated using other means of quantifying expression levels. The goal of this research was to verify regulation of CTR1 and CUP1 using quantitative PCR. RNA was isolated from yeast grown in triplicate under standard conditions with and without 1 mM copper sulfate. Target RNA expression levels were measured by quantitative PCR after conversion of RNA to cDNA and then normalized to the geometric mean of expression levels of three stably expressed reference genes. ANOVA indicated that CTR1 is modestly down- regulated by copper (P=0.101), and CUP1 is significantly up-regulated (P=0.0003). Repression of CTR1 expression by copper is consistent with RNA-Seq results and other studies. Up-regulation of CUP1 was not detected by RNA-Seq but had been observed in other studies. We conclude that RNA-Seq reliably detected regulation of CTR1 but failed to detect regulation of CUP1.

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