scholarly journals Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

2011 ◽  
Vol 56 (No. 5) ◽  
pp. 213-216 ◽  
Author(s):  
M. Nesvadbová ◽  
A. Knoll
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability

The selection of reference genes is essential for gene expression studies when using a real-time quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

scholarly journals Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

2019 ◽  
Vol 70 (4) ◽  
pp. 261-267
Author(s):  
Gaigai Du ◽  
Liyuan Wang ◽  
Huawei Li ◽  
Peng Sun ◽  
Jianmin Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Suitable Reference Gene ◽  
Diospyros Kaki ◽  
Stable Gene ◽  
Expression Studies ◽  
Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

scholarly journals Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

2014 ◽  
Vol 60 (3) ◽  
pp. 451-454 ◽  
Author(s):  
Liesbeth Ceelen ◽  
Jurgen De Craene ◽  
Ward De Spiegelaere
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Heparg Cell ◽  
Expression Studies ◽  
Heparg Cell Line ◽  
Heparg Cells ◽  
Gene Expression Studies

Abstract BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

scholarly journals Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0246227
Author(s):  
Nicole Dittrich Hosni ◽  
Ana Carolina Anauate ◽  
Mirian Aparecida Boim
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
High Glucose ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Expression Studies ◽  
Gene Expression Studies

Background Real-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high-quality qPCR, providing strong support for results, and performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used thus far to enhance the validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g., losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cell and podocyte studies of an in vitro DKD model of disease and its treatment. Methods Five software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted, and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n = 5; 5 mM glucose), mannitol (n = 5; 30 mM, as osmotic control), HG (n = 5; 30 mM glucose), and HG + losartan (n = 5; 30 mM glucose and 10−4 mM losartan). Real-time PCR was performed according to MIQE guidelines. Results We identified that the use of 2 genes was the best combination for qPCR normalization for both mesangial cells and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results. Conclusion This analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR, while for podocytes, HPRT and GAPDH should be chosen.

scholarly journals Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

2021 ◽  
Author(s):  
Nicole Dittrich Hosni ◽  
Ana Carolina Anauate ◽  
Mirian Aparecida Boim
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
High Glucose ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Expression Studies ◽  
Gene Expression Studies

ABSTRACTBackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high quality qPCR, providing strong support for results, as well as performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used so far to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g. Losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cells and podocytes studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n=5; 5mM glucose), mannitol (n=5; 30mM, as osmotic control), HG (n=5; 30mM glucose), and HG + losartan (n=5; 30mM glucose and 10-4 mM of losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes is the best combination for qPCR normalization for both mesangial cell and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR technique, while for podocytes, HPRT and GAPDH should be chosen.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

scholarly journals Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorota M. Krzyżanowska ◽  
Anna Supernat ◽  
Tomasz Maciąg ◽  
Marta Matuszewska ◽  
Sylwia Jafra
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Reference Genes ◽  
Significance Threshold ◽  
Expression Studies ◽  
Reverse Transcription Quantitative Pcr ◽  
Gene Expression Studies ◽  
The Stability ◽  
Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

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