Reference Genes For Expression Studies Research Articles

Reference genes for expression studies in different developmental stages of Oryctes rhinoceros, the coconut rhinoceros beetle

Reference genes for expression studies in different developmental stages of Oryctes rhinoceros, the coconut rhinoceros beetle

I Choose You: Selecting Accurate Reference Genes for qPCR Expression Analysis in Reproductive Tissues in Arabidopsis thaliana

Quantitative real-time polymerase chain reaction (qPCR) is a widely used method to analyse the gene expression pattern in the reproductive tissues along with detecting gene levels in mutant backgrounds. This technique requires stable reference genes to normalise the expression level of target genes. Nonetheless, a considerable number of publications continue to present qPCR results normalised to a single reference gene and, to our knowledge, no comparative evaluation of multiple reference genes has been carried out in specific reproductive tissues of Arabidopsis thaliana. Herein, we assessed the expression stability levels of ten candidate reference genes (UBC9, ACT7, GAPC-2, RCE1, PP2AA3, TUA2, SAC52, YLS8, SAMDC and HIS3.3) in two conditional sets: one across flower development and the other using inflorescences from different genotypes. The stability analysis was performed using the RefFinder tool, which combines four statistical algorithms (geNorm, NormFinder, BestKeeper and the comparative ΔCt method). Our results showed that RCE1, SAC52 and TUA2 had the most stable expression in different flower developmental stages while YLS8, HIS3.3 and ACT7 were the top-ranking reference genes for normalisation in mutant studies. Furthermore, we validated our results by analysing the expression pattern of genes involved in reproduction and examining the expression of these genes in published mutant backgrounds. Overall, we provided a pool of appropriate reference genes for expression studies in reproductive tissues of A. thaliana, which will facilitate further gene expression studies in this context. More importantly, we presented a framework that will promote a consistent and accurate analysis of gene expression in any scientific field. Simultaneously, we highlighted the relevance of clearly defining and describing the experimental conditions associated with qPCR to improve scientific reproducibility.

Selection of Reference Gene for Expression Studies in the Ovary and Pituitary of Spotted Scat (Scatophagus argus) at Different Ovarian Stages

The spotted scat (Scatophagus argus) is the most widely commercially cultured fish in eastern and southern Asia that possess potential economic value. However, up to date, there are no studies reported on the selection of suitable reference genes in this fish, which is crucial for guaranteeing accurate and reliable results in RT-qPCR analyses. This study aimed at screening the most stable reference genes in the ovary and pituitary at different ovarian stages, which is of great significance for further research on the reproductive regulation mechanism of female S. argus. In this study, we selected twenty-one genes as the candidate reference genes, including four genes in the ovary (EEF1A, ACTB, B2M, and HPRT1), and seventeen genes in the pituitary (B2M, APOA1, CGBA, RPLP0, GNRHR, GUSB, TBP, SLC25A5, RPL4, PLA1A, GAPDH, GNB2L1, CTFS, HPRT1, RPS2, TFRC, and TUBB4B). To analyze the stability of the potential reference genes, we first used four commonly used software, BestKeeper, GeNorm, NormFinder, and ΔCt, and then we established comprehensive rankings with ReFinder. The results indicate that RPL4 and GNB2L1, and EEF1A and HPRT1 were the most appropriate reference genes for the pituitary and ovary of S. argus, respectively, in the three developmental stages of the ovary. Additionally, GeNorm analysis suggested that for the accurate normalization of gene expression, two reference genes were reliable in the ovary and pituitary of S. argus. To the best of our knowledge and understanding, this is the first study to identify appropriate reference genes for analyzing gene expression in S. argus. Consequently, this study can be used as a foundation for future research on the reproduction of S. argus.

Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model.

The choice of appropriate reference genes is essential for correctly interpreting qPCR data and results. However, the majority of animal studies use a single reference gene without any prior evaluation. Therefore, many qPCR results from rodent studies can be misleading, affecting not only reproducibility but also translatability. In this study, the expression stability of reference genes for mRNA and miRNA in archived FFPE samples of 117 C57BL/6JOlaHsd mice (males and females) from 9 colitis experiments (dextran sulfate sodium; DSS) were evaluated and their expression analysis was performed. In addition, we investigated whether normalization reduced/neutralized the influence of inter/intra-experimental factors which we systematically included in the study. Two statistical algorithms (NormFinder and Bestkeeper) were used to determine the stability of reference genes. Multivariate analysis was made to evaluate the influence of normalization with different reference genes on target gene expression in regard to inter/intra-experimental factors. Results show that archived FFPE samples are a reliable source of RNA and imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues. Multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes.

Identification of reference genes for expression studies in the liver and spleen of laying hens housed in cage and cage-free systems.

The liver and spleen play a pivotal role in metabolism and immune response. During stress, neuroendocrine response induces changes in gene expression, and its assessment demands the validation of the stability of the reference genes to perform relative gene expression experiments. The objective of this study was to determine the expression stability of four reference genes (GAPDH, ACTB, RNA18S, and HMBS) in the liver and spleen tissues from laying hens housed in a conventional cage (CC) and cage-free (CF) egg production systems. Liver and spleen from Hy-Line Brown hens housed in CC and CF egg production systems were used. mRNA transcript levels were determined by quantitative polymerase chain reaction (qPCR), and the gene expression stability was evaluated using geNorm, BestKeeper, and NormFinder algorithms. The most stable gene from liver tissue was ACTB in CC, CF, and CC-CF groups (overall data). In the spleen, the most stable genes were GAPDH (CC), HMBS (CF), and ACTB (CC-CF). The ACTB gene was the most stable gene in the liver, and GAPDH and HMBS genes were stable in spleen tissues that could be used for the normalization in qPCR experiments performed in liver and spleen tissues of laying hens housed CC and CF production systems.

Identification of stably expressed reference genes for expression studies in Arabidopsis thaliana using mass spectrometry-based label-free quantification.

Arabidopsis thaliana has been used regularly as a model plant in gene expression studies on transcriptional reprogramming upon pathogen infection, such as that by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), or when subjected to stress hormone treatments including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been extensively employed to quantitate these gene expression changes. However, the accuracy of the quantitation is largely dependent on the stability of the expressions of reference genes used for normalization. Recently, RNA sequencing (RNA-seq) has been widely used to mine stably expressed genes for use as references in RT-qPCR. However, the amplification step in RNA-seq creates an intrinsic bias against those genes with relatively low expression levels, and therefore does not provide an accurate quantification of all expressed genes. In this study, we employed mass spectrometry-based label-free quantification (LFQ) in proteomic analyses to identify those proteins with abundances unaffected by Pst DC3000 infection. We verified, using RT-qPCR, that the levels of their corresponding mRNAs were also unaffected by Pst DC3000 infection. Compared to commonly used reference genes for expression studies in A. thaliana upon Pst DC3000 infection, the candidate reference genes reported in this study generally have a higher expression stability. In addition, using RT-qPCR, we verified that the mRNAs of the candidate reference genes were stably expressed upon stress hormone treatments including JA, SA, and ABA. Results indicated that the candidate genes identified here had stable expressions upon these stresses and are suitable to be used as reference genes for RT-qPCR. Among the 18 candidate reference genes reported in this study, many of them had greater expression stability than the commonly used reference genes, such as ACT7, in previous studies. Here, besides proposing more appropriate reference genes for Arabidopsis expression studies, we also demonstrated the capacity of mass spectrometry-based LFQ to quantify protein abundance and the possibility to extend protein expression studies to the transcript level.

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall (scoring highly in whole embryos, heads or forelimbs alone, and in all samples collectively). HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable ( CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5–E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall (scoring highly in whole embryos, heads or forelimbs alone, and in all samples collectively). HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable ( CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BackgroundReal-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates.ResultsIn this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues.ConclusionMytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-022-08553-1.

Identification and validation of stage-specific reference genes for gene expression analysis in Callosobruchus maculatus (Coleoptera: Bruchidae)

In light of a number of recent studies highlighting the increasing research interest in bruchids, it is crucial to validate suitable reference genes that could be used in quantitative gene expression studies. Callosobruchus maculatus is a serious pest of stored grains and field legumes in which reference genes have not been assessed and validated to date. The present study aimed to identify and validate reference genes in different developmental stages of C. maculatus shortlisted from commonly used reference genes such as VATPase, TRIP12, TBP, TF11D, ACTIN, GST, ANNEXIN, PTCD3, RPL32, and β -Tub in various insects. Dedicated algorithms like GeNorm, NormFinder, and BestKeeper were used to analyze the stability of these candidate genes, which revealed GST for third instar, ANNEXIN and PTCD3 for the fourth instar, TF11D and VATPase for male pupa, RPL32 and β-tub for female pupa, β-tub and TBP for adult male and VATPase and GST for adult females as suitable reference genes for expression studies in C. maculatus. The final comprehensive ranking using RefFinder identified GST and TBP as the best reference genes for all the developmental stages of C. maculatus. To the best of our knowledge, this is the first report which evaluates and validates stable reference genes in C. maculatus. The information of stage-specific gene expression, generated in this study will be useful for future molecular, physiological, and biochemical studies on C. maculatus and other closely related bruchids.

Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety.

Simple SummaryReductions in the fertility, body weight, and growth rate of cattle across the world are associated with the global warming phenomenon. Developing optimal management strategies is an important aspect of breeding programs for different breeds. Blood tissue undergoes dramatic physiological and metabolic changes during heat stress conditions, which involves the expression and regulation of a great number of genes across species. Real-time quantitative PCR (qPCR) is a method for the rapid and reliable quantification of mRNA transcription. Reference genes are used to normalize mRNA levels between different samples. Thus, the selection of high-quality reference genes is necessary for the interpretation of data generated by real-time PCR.Real-time PCR is widely used to study the relative abundance of mRNA due to its specificity, sensitivity, and repeatability quantification. However, relative quantification requires a reference gene, which should be stable in its expression, showing lower variation by experimental conditions or tissues. The aim of this study was to evaluate the stability of the expression of five commonly used reference genes (actb, ywhaz, b2m, sdha, and 18s rRNA) at different physiological stages (alert and emergency) in three different cattle breeds. In this study, five genes (actb, ywhaz, b2m, sdha, and 18s rRNA) were selected as candidate reference genes for expression studies in the whole blood from three cattle breeds (Romosinuano, Gyr, and Brahman) under heat stress conditions. The transcription stability of the candidate reference genes was evaluated using geNorm and NormFinder. The results showed that actb, 18SrRNA, and b2m expression were the most stable reference genes for whole blood of Gyr and Brahman breeds under two states of livestock weather safety (alert and emergency). Meanwhile, actb, b2m, and ywhaz were the most stable reference genes for the Romosinuano breed.

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo.

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall. HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable ( CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

Identification of reliable reference genes for expression studies in the magnum of laying hens housed in cage and cage-free systems.

Stress factors during poultry production can evoke changes in gene transcription and protein synthesis in the hen oviduct and could affect the internal and external egg quality. Studies of relative gene expression require the identification of the most stable reference genes for the quantitative polymerase chain reaction (qPCR) to investigate the reproductive tissues' response in laying hens kept in different production systems. The objective of this study was to determine the most stable reference genes of the magnum tissues of laying hens housed in two different production systems. Hy‐Line Brown hens were reared under the same sanitary conditions until 15 weeks of age. Later on, they were transferred into two different production systems, conventional cage (CC) and cage free (CF), up to 82 weeks of age. At 50 and 60 weeks, a total of six hens from each production system were euthanized, and magnum samples were collected. The qPCR was used to determine the RNA transcription level of five reference genes, ACTB, 18S, GAPDH, MSX2 and HMBS. These genes were evaluated for transcript expression in magnum tissues by NormFinder, BestKeeper, geNorm and RefFinder software. The results indicated that the most stable gene in the CF housing system was HMBS in three of the algorithms and in the CC housing system was the 18S, and the best combination of reference genes was HMBS/GAPDH in CF and 18S/HMBS in CC. In conclusion, HMBS, 18S and GAPDH genes could be used together as reference genes for the normalization of the magnum tissues transcript expression of hens in CF and CC housing systems.

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions.

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate.

Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Identification of reliable reference genes for expression studies in maternal reproductive tissues and foetal tissues of pregnant cows.

The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favourable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of 10 potential RGs in maternal reproductive tissues and foetal tissues, and to analyse the effect of RG selection on the calculation of the relative expression of target genes. The expression stability of 10 potential RGs was analysed in eight different tissues from three pregnant dairy cows. Three programs-GeNorm, NormFinder and Bestkeeper-were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable and least stable RGs. The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

Reference Genes for Expression Studies in Human CD8+ Na\xefve and Effector Memory T Cells under Resting and Activating Conditions

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells.

Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Diabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens.

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

Selection of Reference Genes for the Normalization of RT-qPCR Data in Gene Expression Studies in Insects: A Systematic Review.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying expression levels of targeted genes during various biological processes in numerous areas of clinical and biological research. Selection of appropriate reference genes for RT-qPCR normalization is an elementary prerequisite for reliable measurements of gene expression levels. Here, by analyzing datasets published between 2008 and 2017, we summarized the current trends in reference gene selection for insect gene expression studies that employed the most widely used SYBR Green method for RT-qPCR normalization. We curated 90 representative papers, mainly published in 2013–2017, in which a total of 78 insect species were investigated in 100 experiments. Furthermore, top five journals, top 10 frequently used reference genes, and top 10 experimental factors have been determined. The relationships between the numbers of the reference genes, experimental factors, analysis tools on the one hand and publication date (year) on the other hand was investigated by linear regression. We found that the more recently the paper was published, the more experimental factors it tended to explore, and more analysis tools it used. However, linear regression analysis did not reveal a significant correlation between the number of reference genes and the study publication date. Taken together, this meta-analysis will be of great help to researchers that plan gene expression studies in insects, especially the non-model ones, as it provides a summary of appropriate reference genes for expression studies, considers the optimal number of reference genes, and reviews the average number of experimental factors and analysis tools per study.