Selection of reference genes for gene expression studies in astrocytomas

Gresner, Sylwia M.; Golanska, Ewa; Kulczycka-Wojdala, Dominika; Jaskólski, Dariusz J.; Papierz, W; Liberski, Paweł P.

doi:10.1016/j.ab.2010.09.010

Cited by 16 publications

(12 citation statements)

References 13 publications

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“…Data are expressed as the SCD:GAPDH ratio. Although stability of GAPDH was not determined in this study, recent studies in human diploid fibroblasts (Zainuddin et al, 2010) and astocytomas (Gresner et al, 2011) demonstrated that GAPDH mRNA expression was stable under those conditions.…”

Section: Preparation and Analysis Of Rnamentioning

confidence: 71%

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers1

Brooks

Choi

Lunt

et al. 2011

Journal of Animal Science

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We proposed that stearoyl-CoA desaturase (SCD) activity dictates fatty acid composition of adipose tissue and muscle in beef cattle, regardless of ruminal or hepatic fatty acid hydrogenation or desaturation. Twelve Angus steers were assigned to a calf-fed (CF) group and slaughtered at weaning (8 mo of age; n=4), 12 mo of age (n=4), or 16 mo of age (n=4). Twelve steers were assigned to a yearling-fed (YF) group and slaughtered at 12 mo of age (n=4), 16 mo of age (n=4), and 17.5 mo of age (n=4; 525 kg, market weight). Data were analyzed based on time on the corn-based finishing diet, with terminal age as a covariate, and orthogonal polynomial contrasts were tested on the main effects of treatment group and time on the finishing diet. Fatty acids from duodenal digesta, plasma, liver, LM, and subcutaneous and intramuscular adipose tissue were measured, and SCD gene expression was measured in intramuscular and subcutaneous adipose tissues. In duodenal digesta, palmitic and linoleic acids increased by 100% over the sampling period, α-linolenic acid decreased over the sampling period, and trans-vaccenic acid was greater in YF than in CF steers (all P < 0.01). The proportion of α-linolenic acid decreased over time in all tissues, including liver. The SCD index (ratio of SCD fatty acid products to SCD fatty acid substrates) increased over time in LM and in intramuscular and subcutaneous adipose tissues. The SCD:glyceraldehyde 3-phosphate dehydrogenase mRNA ratio was virtually undetectable at the initial sampling periods in subcutaneous adipose tissue of YF and CF steers, and it increased over time (P < 0.01). The SCD index and SCD:glyceraldehyde 3-phosphate dehydrogenase ratio were greater in intramuscular adipose tissue of CF steers than in that of YF steers. The SCD index did not change over time in liver and decreased over time in duodenal digesta. We conclude that, unlike essential fatty acids, the SFA and MUFA composition of adipose tissue is regulated by adipose tissue fatty acid desaturation, with little contribution from hepatic or duodenal fatty acids.

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Section: Preparation and Analysis Of Rnamentioning

confidence: 71%

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers1

Brooks

Choi

Lunt

et al. 2011

Journal of Animal Science

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“…An ideal RG should be neither influenced nor regulated by experimental conditions or treatments. Increasing evidence indicated that there is no single RG that is able to be used for multiple experiments; however, an increasing number of studies suggested that a group of putative RGs for certain specific experimental setups may be recommended for future studies (35)(36)(37). MSCs, primarily derived from BM, have been examined widely for their capacities in repairing damaged tissues (38)(39)(40).…”

Section: Discussionmentioning

confidence: 99%

Identification of optimal reference genes for quantitative PCR studies on human mesenchymal stem cells

Yang

Bai

et al. 2014

Molecular Medicine Reports

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Quantitative polymerase chain reaction (qPCR) analysis is a commonly used method for the study of mRNA expression throughout the field of mesenchymal stem cell (MSC) research. This technology is simple and sensitive; however the results may vary significantly due to the use of various reference genes (RGs) as normalizers. Therefore, the reliable use of RGs is vital for obtaining accurate results. The present study focuses on ten putative RGs for the normalization of qPCR data between human bone marrow-derived MSCs (BM-MSCs) and fetal tissue-derived MSCs (FT-MSCs). The total RNA from these two types of MSC was isolated using TRIzol reagent. cDNA was generated from the RNA via reverse transcription and subsequently analyzed by qPCR using ten common RGs as normalizers. These RGs included 18S, ACTB, B2M, HPRT1, GAPDH, TBP, PPIA, RPLP0, PGK1 and RPL13A. GeNorm, NormFinder and BestKeeper software were used to analyze the qPCR results by evaluating the expression stabilities of the ten candidate RGs in BM-MSCs and FT-MSCs. Consequently, several of the commonly used RGs, including 18S, ACTB and TBP, were demonstrated to be unsuitable for normalization in these two MSCs, whereas RPL13A, B2M and PPIA were the most stable RGs and were therefore reliable for use in qPCR studies. Combining multiple RGs had no contribution towards increasing their stabilities. In conclusion, the present study revealed that RPL13A, B2M and PPIA were the optimal RGs for qPCR studies comparing BM-MSCs and FT-MSCs.

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“…When a gene of interest is not compared to appropriately validated, stably expressed reference genes, misinterpretation of results may occur. Constantly growing evidence indicates that there is no single reference gene that can be used for different experiments, but hopefully with the growing number of experimental data and reports, such as this one, a group of putative reference genes for certain specific experimental setups could be recommended for future studies [5], [6], [19], [20].…”

Section: Discussionmentioning

confidence: 99%

“…There are several strategies which can be applied to normalize qPCR results [3], but the most common one is the use of reference genes as an internal standard. Although recent studies clearly show the importance of a proper choice of a reference gene [2], [5], [6], still many currently published reports present RT-qPCR results that miss information on a reference gene selection. In addition, researchers often routinely use the most classical reference genes, such as genes coding for glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) or β-actin ( ACTB ), convinced that these are the universal reference genes and unaware that they can be highly regulated [7]–[9].…”

Section: Introductionmentioning

confidence: 99%

Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells

Żyżyńska-Granica

Koziak

2012

PLoS ONE

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Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that β-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.

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Selection of reference genes for gene expression studies in astrocytomas

Cited by 16 publications

References 13 publications

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers1

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers1

Identification of optimal reference genes for quantitative PCR studies on human mesenchymal stem cells

Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells

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