Quantitative analysis of gene expression relative to 18S rRNA in carcinoma samples using the LightCycler instrument and a SYBR GreenI-based assay: determining FAT10 mRNA levels in hepatocellular carcinoma.

Abstract

Due to the fact that mutations and up- or downregulation of genes can lead to the development of cancer, quantitative comparison of relative gene expression in healthy and cancerous tissue can gain valuable insights into tumorigenesis. While the semi-quantitative DNA microarrays are being used to identify differentially expressed genes on a genomic scale, real-time RT-PCR provides a powerful tool for quantitative measurement of gene expression. Presently, it is the most sensitive method available. Here we describe in detail a SYBR GreenI-based assay using the LightCycler instrument to measure the levels of mRNA for the ubiquitin-like protein FAT10 relative to 18S rRNA in human hepatocellular carcinoma tissue. This method can be easily adapted to any tissue (human or mouse, rat, etc.) and any gene.