Stable reference genes for RT-qPCR analysis of gene expression in the Musa acuminata-Pseudocercospora musae interaction

Abstract Leaf pathogens are limiting factors in banana (Musa spp.) production, with Pseudocercospora spp. responsible for the important Sigatoka disease complex. In order to investigate cellular processes and genes involved in host defence responses, quantitative real-time PCR (RT-qPCR) is an analytical technique for gene expression quantification. Reliable RT-qPCR data, however, requires that reference genes for normalization of mRNA levels in samples are validated under the conditions employed for expression analysis of target genes. We evaluated the stability of potential reference genes ACT1, α-TUB, UBQ1, UBQ2, GAPDH, EF1α, APT and RAN. Total RNA was extracted from leaf tissues of Musa acuminata genotypes Calcutta 4 (resistant) and Cavendish Grande Naine (susceptible), both subjected to P. musae infection. Expression stability was determined with NormFinder, BestKeeper, geNorm and RefFinder algorithms. UBQ2 and RAN were the most stable across all M. acuminata samples, whereas when considering inoculated and non-inoculated leaf samples, APT and UBQ2 were appropriate for normalization in Calcutta 4, with RAN and α-TUB most stable in Cavendish Grande Naine. This first study of reference genes for relative quantification of target gene expression in the M. acuminata-P. musae interaction will enable reliable analysis of gene expression in this pathosystem, benefiting elucidation of disease resistance mechanisms.

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Optimizations for identifying reference genes in bone and cartilage bioengineering

BMC Biotechnology ◽

10.1186/s12896-021-00685-8 ◽

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.

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Identification and Validation of Reference Genes for Gene Expression Analysis in Different Development Stages of Amylostereum areolatum

Frontiers in Microbiology ◽

10.3389/fmicb.2021.827241 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ningning Fu ◽

Jiaxing Li ◽

Ming Wang ◽

Lili Ren ◽

Shixiang Zong ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Growth And Development ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Growth Stages ◽

Experimental Conditions ◽

Development Stages ◽

Symbiotic Fungus ◽

The Stability

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

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Optimizations for Identifying Reference Genes in Bone and Cartilage Bioengineering

10.21203/rs.3.rs-96393/v1 ◽

2020 ◽

Author(s):

Fei Xiong ◽

Xiangyun Cheng ◽

Chao Zhang ◽

Roland Manfred Klar ◽

Tao He

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Adipose Tissue ◽

Reference Gene ◽

Muscle Tissue ◽

Reference Genes ◽

Target Genes ◽

Gene Set ◽

The Stability ◽

And Cartilage

Abstract Background：Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly considered as the best-established technique for gene expression assay. However, appropriate reference gene set selection remains the critical and challenging subject for the proper understanding of gene expression pattern. Mixed opinions pertain in how to choose optimal reference gene set according 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 was the most feasible for the identification of reference genes in bone and cartilage bioengineering experiments. 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 ensuring the 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 different schemes may 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 and eventual normalization of target genes showed that the different selection schemes have significant effect on the eventual normalization of target genes.Conclusions：Based on the results, the proposed cut-off value of Vn/n+1 under 0.15, according to geNorm algorithm, should be considered with caution, especially in certain tissue types such as skeletal muscle and adipose tissue. Instead, the minimum Vn/n+1 should be used as the cut-off for choosing the optimal reference gene set especially when the stability and variation of candidate reference genes in a specific study are unclear.

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Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

Research Society and Development ◽

10.33448/rsd-v9i11.9702 ◽

2020 ◽

Vol 9 (11) ◽

pp. e1599119702

Author(s):

Marina Martins de Oliveira ◽

Kalynka Gabriella do Livramento ◽

Maísa Lamounier Magalhães ◽

Luciano Vilela Paiva ◽

Ana Paula Peconick

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Target Gene ◽

Target Genes ◽

Cardiac Tissue ◽

Rna Extraction ◽

Obesity And Diabetes ◽

The Stability ◽

Universal Reference

The validation of reference genes is an essential step for any RT-qPCR analysis. In this way, the present paper aimed to identify and validate reference genes for RT-qPCR in cardiac tissue of rats of the Rattus norvegicus albinus specie, submitted to obesity associated or not to type 2 diabetes mellitus. For this, the metabolic changes were induced at the 42nd day of life and the euthanasia was performed on the 70th day. The heart apexes were collected and destinates for RNA extraction. The RT-qPCR technique was performed in own thermocycler, the efficiency of the primers found by the LinReg software and the stability of the expression of the reference genes in the samples was analyzed by the RefFinder algorithm. The candidates for reference genes were GAPDH, POLR2A, RPL32, and RPL4 and the target gene used to verify the differences in gene expression of candidates for reference genes was CMA1. The obese animals showed a decrease in CMA1 gene expression when compared to the two most stable reference genes. The opposite occurs when it is compared to the two less stable reference genes. The GAPDH and POLR2A genes are the best to normalize the reactions with the samples in question. There is no universal reference gene for all situations, which requires systematic validation for each situation. The use of unvalidated reference genes may compromise the interpretation of the expression of the target genes, which would prevent the reflection of the actual situation.

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Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues – a multistudy analysis

BMC Veterinary Research ◽

10.1186/s12917-020-02635-6 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Marta Nowak ◽

Selim Aslan ◽

Mariusz P. Kowalewski

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Large Scale ◽

Target Genes ◽

Reproductive Tract ◽

Morphological Changes ◽

Reproductive Organs ◽

Potential Candidate ◽

Altered Gene ◽

The Stability

Abstract Background Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other species, the reproductive tract is continuously undergoing hormonal and cycle stage-dependent morphological changes, which are associated with altered gene expression. However, there have been few attempts published so far targeted to the dog aimed at determining optimal reference genes for the reproductive organs. Most of these approaches relied on genes previously described in other species. Large-scale transcriptome-based experiments are promising tools for defining potential candidate reference genes, but were never considered in this context in canine research. Results Here, using available microarray and RNA-seq datasets derived from reproductive organs (corpus luteum, placenta, healthy and diseased uteri) of dogs, we have performed multistudy analysis to identify the most stably expressed genes for expression studies, in each tissue separately and collectively for different tissues. The stability of newly identified reference genes (EIF4H, KDELR2, KDM4A and PTK2) has been determined and ranked relative to previously used reference genes, i.e., GAPDH, β-actin and cyclophillin A/PPIA, using RefFinder and NormFinder algorithms. Finally, expression of selected target genes (luteal IL-1b and MHCII, placental COX2 and VEGFA, and uterine IGF2 and LHR) was re-evaluated and normalized. All proposed candidate reference genes were more stable, ranked higher and introduced less variation than previously used genes. Conclusions Based on our analyses, we recommend applying KDM4A and PTK2 for normalization of gene expression in the canine CL and placenta. The inclusion of a third reference gene, EIF4H, is suggested for healthy uteri. With this, the interpretation of qPCR data will be more reliable, allowing better understanding of canine reproductive physiology.

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Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.)

Scientific Reports ◽

10.1038/s41598-021-83918-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Paulo H. da Silva Santos ◽

João R. Vieira Manechini ◽

Michael S. Brito ◽

Elisson Romanel ◽

Renato Vicentini ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Floral Induction ◽

Photoperiodic Induction ◽

Experimental Conditions ◽

Wide Range ◽

The Stability ◽

Stability And Consistency

AbstractAlthough reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.

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Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Scientific Reports ◽

10.1038/s41598-020-79298-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuping Li ◽

Xiaoju Liang ◽

Xuguo Zhou ◽

Yu An ◽

Ming Li ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stage ◽

Reference Genes ◽

Developmental Stages ◽

Individual Factors ◽

Congeneric Species ◽

Spatio Temporal ◽

The Stability ◽

Different Tissues ◽

Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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

Genes ◽

10.3390/genes12070960 ◽

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.

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Identification of stable reference genes in differentiating human pluripotent stem cells

Physiological Genomics ◽

10.1152/physiolgenomics.00130.2014 ◽

2015 ◽

Vol 47 (6) ◽

pp. 232-239 ◽

Cited By ~ 11

Author(s):

Gustav Holmgren ◽

Nidal Ghosheh ◽

Xianmin Zeng ◽

Yalda Bogestål ◽

Peter Sartipy ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Expression Data ◽

Pluripotent Stem Cells ◽

Reference Genes ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Expression Data ◽

Large Variability ◽

The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

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

Scientific Reports ◽

10.1038/s41598-019-49474-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

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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