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Selection of reference genes for gene expression studies in human bladder cancer using SYBR-Green quantitative polymerase chain reaction.

ABSTRACT: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a rapid, reliable and widely used method of studying gene expression profiles that requires appropriate normalization for accurate and reliable results. Reference genes are usually used to normalize mRNA levels; however, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions. Therefore, a normalization strategy is an important precondition for reliable conclusions, with endogenous controls requiring determination for every experimental system. In the present study, 18 reference genes used in various prior studies were analyzed to determine their applicability in bladder cancer. A total of 35 matched malignant and non-malignant bladder cancer (specifically transitional cell carcinoma) tissue specimens were examined. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed using SYBR-Green RT-qPCR. mRNA abundance was compared and reference genes with distinct ranges of expression to possible target genes were excluded. Genes that were differentially expressed in matched non-cancerous and cancerous samples were also excluded, using quantification cycle analysis. Subsequently, the stability of the selected reference genes was analyzed using three different methods: geNorm, NormFinder and BestKeeper. The rarely used ribosomal protein S23 (RPS23) was the most stable single reference gene, with RPS23, tumor protein, translationally controlled 1 and RPS13 comprising the optimal reference gene set for all the bladder samples. These stable reference genes should be employed in normalization and quantification of transcript levels in future expression studies of bladder cancer-associated genes.

SUBMITTER: Zhang C

PROVIDER: S-EPMC5661485 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods:Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-?, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) less than 0.2 and coefficient of variation (CV-value) less than 0.1. Results:Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11-21 (41-78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions:We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.

Project description:BACKGROUND: Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut. RESULTS: A set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied. CONCLUSIONS: This first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.

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Selection of reference genes for gene expression studies in human bladder cancer using SYBR-Green quantitative polymerase chain reaction.

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