Rabbit Anti-PPIA monoclonal antibody, clone KN218-14

Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. Asystematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqManw array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitinC(UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the studywas validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNAexpression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIAwas identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.

The stability of expression of an internal control is required for accurate and reliable normalization in quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) experiments. However, expression of commonly used reference genes can be regulated under specific experimental conditions, particularly in response to stimuli that exert multiple effects on gene expression. The neurotrophin brain-derived neurotrophic factor (BDNF) regulates gene expression through activation of multiple signaling cascades, and we have conducted an expression study for the proper validation of internal control genes in BDNF-stimulated cultured hippocampal neurons. geNorm and NormFinder were applied to eight potential genes to identify the most stable genes to be used in the relative quantification of the effects of BDNF on gene expression. Our data show that Tbp (TATA box binding protein), Ppia (peptidylprolyl isomerase A), Pgk1 (phosphoglycerate kinase 1), and Hprt1 (hypoxanthine guanine phosphoribosyl transferase 1) are the most stable genes under the experimental conditions used, contrasting with Tubal (tubulin alphal-A chain) and Gapdh (glyceraldehydes-3-phosphate dehydrogenase), two genes widely used as control genes, which showed an unstable expression in hippocampal neurons stimulated with BDNF. Analysis of the BDNF-induced changes in expression of Sars, Tufm, and Egr3 by using different sets of control genes showed distinct results, with a combination of three to four of the genes Tbp/Ppia/Pgk1/Hprt1 providing the most consistent results. Our data reinforce the need for proper validation of the internal control genes for an accurate quantification of qRT-PCR results, particularly when analyzing cellular responses to agents (e.g., neurotrophins) that cause multiple changes in gene expression. (c) 2008 Wiley-Liss, Inc.