Identification of endogenous control genes for normalisation of real-time quantitative PCR data in colorectal cancer

Abstract

BackgroundGene expression analysis has many applications in cancer diagnosis, prognosis and therapeutic care. Relative quantification is the most widely adopted approach whereby quantification of gene expression is normalised relative to an endogenously expressed control (EC) gene. Central to the reliable determination of gene expression is the choice of control gene. The purpose of this study was to evaluate a panel of candidate EC genes from which to identify the most stably expressed gene(s) to normalise RQ-PCR data derived from primary colorectal cancer tissue.ResultsThe expression of thirteen candidate EC genes: B2M, HPRT, GAPDH, ACTB, PPIA, HCRT, SLC25A23, DTX3, APOC4, RTDR1, KRTAP12-3, CHRNB4 and MRPL19 were analysed in a cohort of 64 colorectal tumours and tumour associated normal specimens. CXCL12, FABP1, MUC2 and PDCD4 genes were chosen as target genes against which a comparison of the effect of each EC gene on gene expression could be determined. Data analysis using descriptive statistics, geNorm, NormFinder and qBasePlus indicated significant difference in variances between candidate EC genes. We determined that two genes were required for optimal normalisation and identified B2M and PPIA as the most stably expressed and reliable EC genes.ConclusionThis study identified that the combination of two EC genes (B2M and PPIA) more accurately normalised RQ-PCR data in colorectal tissue. Although these control genes might not be optimal for use in other cancer studies, the approach described herein could serve as a template for the identification of valid ECs in other cancer types.

Elevated Expression of Hypoxanthine Guanine Phosphoribosyltransferase within Malignant Tissue

“…After comparing various housekeeping genes such as GAPDH, β-2 microglobulin, 18s ribosomal RNA, etc., some researchers have reported HPRT as the most consistent endogenous control (de Kok et al, 2005). Meanwhile, other researchers have reported HPRT levels to be significantly lower than other controls in cancer tissue (Kheirelseid, Chang, Newell, Kerin, & Miller, 2010). Finally, other studies have reported HPRT as an unsuitable standard in certain cell types due to varying expression in response to growth factor stimuli (Murphy & Polak, 2002).…”

Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues

“…These include 5 microRNAs (miR-23a, miR-200c, miR-26a, miR-1979, and let7a) which were chosen based on a cervical microRNA microarray expression data, two microRNAs (miR-191 and miR-103) which had be reported to be the most stable reference RNA targets in microRNA RT-qPCR studies (Peltier and Latham, 2008), 5S rRNA (121 nt) and U6 snRNA owing to their purported expression stability and use in several published microRNA RT-qPCR studies (Vandesompele et al, 2002;Peltier and Latham, 2008;Kheirelseid et al, 2010). We filtered the microRNA microarray expression data (form 6 HR-HPV negative cervical normal tissues and 6 HR-HPV positive cancer tissues) according to three criteria: (1) microRNA must be highly expressed in most of the samples; (2) microRNA must be consistently expressed (measured by the modified z-score), with a small coefficient of variation (CV); (3) only one representative microRNA was considered from a given family.…”

MicroRNA-21 and PDCD4 expression in colorectal cancer

“…PDCD4 expression levels were normalised to PPIA and β2M expression levels [25]. Percent PCR amplification efficiencies (E) for each assay were calculated as E = (10-1/slope -1) × 100, using the slope of the semi-log regression plot of Ct versus log input of cDNA (10-fold dilution series of five points) A threshold of 10% above or below 100% efficiency was applied.…”