Recent reports, however, claim that stably expressed genes in one

Recent reports, however, claim that stably expressed genes in one tumour type may not predict stable expression in another tumour type [12, 27]. Moreover, results in one tumour type, like colorectal cancer, show stably expressed genes in one experimental in which are different from the stably

expressed genes in another experimental setup [28–30]. Hence, reference genes should be validated and selected in every experiment in any tissue type. Recently, it has been suggested that the focus should be on introducing and validating novel approach for reference gene identification and standardizing experimental setup rather than giving general suggestions for different tissues [16]. Applying TaqMan Low Density Array (TLDA) to examining reference genes is a step towards a more standardized experimental setup. TLDA was evaluated in colorectal FHPI cell line cancer by Lü Selleckchem Buparlisib et al., 2008, as a roughly robust and labour-saving method for gene quantification compared with routine qRT-PCR [31]. Well-designed TaqMan probes require little optimization, and TLDA allows simultaneously real-time detection of many gene products in several samples offering higher through put than established single array method [31, 32]. Hence, in the present study we used TLDA to find potential reference genes for data normalization in qRT-PCR experiments in metastatic and

non-metastatic colon cancer patients. The gene expression of 16 commonly used reference genes in tumour tissue and individual-matched normal mucosa of metastatic and non-metastatic colon cancer patients were analyzed and the expression stability was determined and compared using geNorm and NormFinder. Methods KU55933 mouse Patients and tissue specimens RNAlater-stored tumour tissue samples and individual-matched normal mucosa were obtained from 38 patients with colonic adenocarcinoma who underwent resection at Akershus University Hospital Tenofovir purchase Trust between 2004 and 2009. The dissected tissue samples were collected in the operating room and stored immediately in approximately five

volumes of RNAlater (Ambion Inc., Austin TX, USA) and frozen at -80°C. Eighteen patients with non-metastatic disease, Dukes B (with a minimum of 12 negative lymph nodes) where no metastases occurred during 5 years follow up, and 20 patients originally staged as Duke C who displayed distant metastases during a 5 year follow-up (Duke C) or patients classified as Dukes D were included in the study. There were 22 women and 16 men with a mean age of 69 +/- 14 years (range 29-92) at surgery. Three sectioned pieces of the tumour samples were made. The central piece was further processed for RNA isolation, while the two end pieces were fixed in formalin and embedded in paraffin (FFPE). Four μm sections of FFPE samples were stained with Hagens Hematoxylin and examined by a pathologist for determination of percentage tumour cells. To avoid bias from necrosis or minimal tumour representation we included tumour tissue samples with more than 70% tumour cells.

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