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.
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.
Lukasiak
Sebastian
S
Department of Biology, University of Konstanz Universitätsstr, Konstanz, Germany.
Breuhahn
Kai
K
Schiller
Claudia
C
Schmidtke
Gunter
G
Groettrup
Marcus
M
D052160:Benzothiazoles; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D003959:Diamines; D015870:Gene Expression; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014452:Ubiquitins / Q000235:genetics*
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; C109184:UBD protein, human; D014452:Ubiquitins; C098022:SYBR Green I