Assessment of housekeeping genes for use in normalization of real time PCR in skeletal muscle with chronic degenerative changes

Yüzbaşıoğlu, Ayşe; Onbaşılar, İlyas; Kocaefe, Çetin; Özgüç, Meral

doi:10.1016/j.yexmp.2009.12.007

Cited by 31 publications

(26 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As suggested by several studies, the accuracy of qRT‐PCR can be improved by using more than one RG (Vandesompele et al., 2002; Yüzbaşıoğlu, Onbaşılar, Kocaefe, & Özgüç, 2010). The optimal number of NFs for normalization was evaluated by pairwise variation analysis using geNorm.…”

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Zhang

Wang

et al. 2017

Ecology and Evolution

View full text Add to dashboard Cite

Changes in gene expression patterns can reflect the adaptation of organisms to divergent environments. Quantitative real‐time PCR (qRT‐PCR) is an important tool for ecological adaptation studies at the gene expression level. The quality of the results of qRT‐PCR analysis largely depends on the availability of reliable reference genes (RGs). To date, reliable RGs have not been determined for adaptive evolution studies in insects using a standard approach. Here, we evaluated the reliability of 17 candidate RGs for five Gynaephora populations inhabiting various altitudes of the Tibetan Plateau (TP) using four independent (geNorm, NormFinder, BestKeeper, and the deltaCt method) and one comprehensive (RefFinder) algorithms. Our results showed that EF1‐α, RPS15, and RPS13 were the top three most suitable RGs, and a combination of these three RGs was the most optimal for normalization. Conversely, RPS2,ACT, and RPL27 were the most unstable RGs. The expression profiles of two target genes (HSP70 and HSP90) were used to confirm the reliability of the chosen RGs. Additionally, the expression patterns of four other genes (GPI,HIF1A,HSP20, and USP) associated with adaptation to extreme environments were assessed to explore the adaptive mechanisms of TP Gynaephora species to divergent environments. Each of these six target genes showed discrepant expression patterns among the five populations, suggesting that the observed expression differences may be associated with the local adaptation of Gynaephora to divergent altitudinal environments. This study is a useful resource for studying the adaptive evolution of TP Gynaephora to divergent environments using qRT‐PCR, and it also acts as a guide for selecting suitable RGs for ecological and evolutionary studies in insects.

show abstract

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Zhang

Wang

et al. 2017

Ecology and Evolution

View full text Add to dashboard Cite

show abstract

“…In each case, controls were performed to ensure expression levels were from cDNA and not contaminating genomic DNA and that each primer pair only amplified the target Tbx gene (as determined by testing each primer pair on plasmids for each Tbx gene). Actin was picked to standardize the amount of expression calculated for Tbx genes at each developmental stage because it has often been utilized in other systems (including cnidarians) for this purpose (McCurley and Callard 2008;Rodriguez-Lanetty et al 2008;Yüzbaşıoğlu et al 2010). Nonetheless, we used qRT-PCR to compare actin mRNA levels at each developmental stage compared to total RNA amounts and though some degree of variability was observed, the expression stability across larval developmental stages was high.…”

Section: Expression Analysesmentioning

confidence: 99%

Expansion, diversification, and expression of T-box family genes in Porifera

et al. 2010

View full text Add to dashboard Cite

Sponges are among the earliest diverging lineage within the metazoan phyla. Although their adult morphology is distinctive, at several stages of development, they possess characteristics found in more complex animals. The T-box family of transcription factors is an evolutionarily ancient gene family known to be involved in the development of structures derived from all germ layers in the bilaterian animals. There is an incomplete understanding of the role that T-box transcription factors play in normal sponge development or whether developmental pathways using the T-box family share similarities between parazoan and eumetazoan animals. To address these questions, we present data that identify several important T-box genes in marine and freshwater sponges, place these genes in a phylogenetic context, and reveal patterns in how these genes are expressed in developing sponges. Phylogenetic analyses demonstrate that sponges have members of at least two of the five T-box subfamilies (Brachyury and Tbx2/3/4/5) and that the T-box genes expanded and diverged in the poriferan lineage. Our analysis of signature residues in the sponge T-box genes calls into question whether "true" Brachyury genes are found in the Porifera. Expression for a subset of the T-box genes was elucidated in larvae from the marine demosponge, Halichondria bowerbanki. Our results show that sponges regulate the timing and specificity of gene expression for T-box orthologs across larval developmental stages. In situ hybridization reveals distinct, yet sometimes overlapping expression of particular T-box genes in free-swimming larvae. Our results provide a comparative framework from which we can gain insights into the evolution of developmentally important pathways.

show abstract

“…Therefore, in this study, we evaluated the most suitable housekeeping gene for RNA expression analysis in normoxic, hypoxic and PHDI-preconditioned CDCs isolated from both neonatal and adult rat hearts, using a panel of 6 housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp) (Table 1). These genes were chosen as they are most commonly used as reference genes in rat qRT-PCR studies [16, 22–32] (Additional file 1).…”

Section: Introductionmentioning

confidence: 99%

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

et al. 2011

View full text Add to dashboard Cite

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-011-1281-5) contains supplementary material, which is available to authorized users.

show abstract

Assessment of housekeeping genes for use in normalization of real time PCR in skeletal muscle with chronic degenerative changes

Cited by 31 publications

References 14 publications

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Expansion, diversification, and expression of T-box family genes in Porifera

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

Contact Info

Product

Resources

About