Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer.
BACKGROUND
Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification due to its methodological reproducibility and sensitivity. The gene expression is quantified by normalization to one or more reference genes which are presumed stably expressed throughout a given experiment. The aim of this study was to validate a standardized experimental setup to identifying reference genes for normalization of qRT-PCR in the metastatic and non-metastatic colon cancer.


METHODS
In this study, expression of 16 commonly used reference genes was quantified in tumour tissue and individual-matched normal mucosa in 18 non-metastatic colon cancer patients and 20 colon cancer patients with distant metastases using TaqMan Low Density Array (TLDA). The expression stability was determined and compared by means of geNorm and NormFinder.


RESULTS
Two pairs of genes, HPRT1/PPIA and IPO8/PPIA, were identified to be suitable to normalize gene expression data in metastatic and non-metastatic colon cancer patients, according to geNorm and NormFinder respectively.


CONCLUSIONS
We propose a standardized approach of finding the most suitable reference gene(s) in every qRT-PCR experiment using TLDA.
Quality and Research Department, Ostfold Hospital Trust, 1603 Fredrikstad, Norway. l.a.sorby@medisin.uio.no
Lise Aa
LA
Sørby
Solveig N
SN
Andersen
Ida R K
IR
Bukholm
Morten B
MB
Jacobsen
D000230:Adenocarcinoma / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003110:Colonic Neoplasms / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000592:standards*; D006801:Humans; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D012333:RNA, Messenger