Combination Of Reference Genes Research Articles

Identification and Evaluation of qRT-PCR Reference Genes in Melanaphis sacchari.

Appropriate reference genes must be selected for accurate qRT-PCR data to conduct a thorough gene expression analysis in the sorghum aphid (Melanaphis sacchari, Hemiptera, Aphididae). This approach will establish a foundation for gene expression analysis and determines the efficacy of RNA interference in the sorghum aphid. Nine potential reference genes, including Actin, 18S, GAPDH, RPL7, EF-1α, EF-1β, 28S, HSP70, and TATA, were assessed under various experimental conditions to gauge their suitability based on qRT-PCR Ct values. The stability of these candidate reference genes in diverse experimental setups was analyzed employing several techniques, including the ΔCt comparative method, geNorm, Normfinder, BestKeeper, and RefFinder. The findings revealed that the quantity of ideal reference genes ascertained by the geNorm method for diverse experimental conditions remained consistent. For the developmental stages of the sorghum aphid, RPL7 and 18S proved to be the most dependable reference genes, whereas GAPDH and EF-1β were recommended as the most stable reference genes for different tissues. In experiments involving wing dimorphism, EF-1α and GAPDH were identified as the optimal reference gene pair. Under varying temperatures, EF-1α and EF-1β were found to be the most dependable gene pair. For studies focusing on insecticide susceptibility, 18S and TATA emerged as the most stable candidate reference genes. Across all experimental conditions, EF-1α and EF-1β was the optimal combination of reference genes in the sorghum aphid. This research has pinpointed stable reference genes that can be utilized across various treatments, thereby enhancing gene expression studies and functional genomics research on the sorghum aphid.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanuserythropterus

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanuserythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L.erythropterus.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae).

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.

Epigenetic-related transcriptional reprogramming elucidated by identification and validation of a novel reference gene combination for RT-qPCR studies in porcine oocytes of contrasting quality

BackgroundReliable RT-qPCR results are dependent on appropriate normalisation. Oocyte maturation studies can be challenging in this respect, as the stage of development can distinctively affect reference gene transcript abundance. The aim of this study was to validate the use of reference genes in oocyte in vitro maturation RT-qPCR studies, and thereafter, examine the abundance of transcripts supporting histone modification during oocyte and early embryo development in oocytes of contrasting quality.Methods and resultsTotal RNA from oocytes from prepubertal gilts and sows was extracted either directly succeeding follicle aspiration or after 44 h in vitro maturation, followed by RT-qPCR. The stability of YWHAG, HPRT1, ACTB, GAPDH, HMBS and PFKP, was analysed by NormFinder and further cross-validated by assessing results generated following application of different combinations of potential reference genes for normalisation of the RT-qPCR data. Combining ACTB and PFKP generated high stability according to NormFinder and concordant results. Applying this normalisation, gilt derived oocytes displayed significantly higher abundance than oocytes from sows of almost all the epigenetic-related transcripts studied (HDAC2, SIRT1, SALL4, KDM1A, KDM1B, KDM5A), both before and after maturation.ConclusionsThis study identified the combined use of ACTB and PFKP as the optimal normalisation for porcine oocyte RT-qPCR data. In oocytes collected from prepubertal gilts, transcription did not appear to be silenced at the time of aspiration, and accumulation of transcripts supporting histone modification facilitating proper fertilization and further embryo development seemed delayed. The results imply the epigenetic-related transcripts may have potential as markers of oocyte quality.

Screening and validation of optimal miRNA reference genes in different developing stages and tissues of Lilium henryi Baker

Dynamic miRNA detection using the qRT-PCR technique requires appropriate reference genes to ensure data reliability. Previous studies have screened internal reference genes in plants during embryonic development and various stress treatment, involving relatively few tissues and organs. There is no relevant miRNA study in Lilium henryi Baker and limited research on the optimal miRNA reference genes in lilies, such as 5S, 18S, U6 and Actin. Twelve genes were selected as candidate reference genes whose expression stability was analyzed in petals at different developmental stages and other tissues using various algorithms, such as geNorm, NormFinder, BestKeeper, and Delta CT. The results revealed that the optimal combination of reference genes for Lilium henryi Baker petals at different developmental stages was osa-miR166m and osa-miR166a-3p, while that for different tissues of Lilium henryi Baker was osa-miR166g-3p and osa-miR166a-3p.Four important genes related to growth and development regulation, namely, osa-miR156a, osa-miR395b, osa-miR396a-3p, and osa-miR396a-5p, were selected for validation. The findings of the present study could contribute to future investigations onmiRNA expression and the related functions in Lilium henryi Baker while providing important references for the normalization of the miRNA expression in other varieties of lily.

Transcriptome-Based Identification of the Optimal Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Analyses of Lingonberry Fruits throughout the Growth Cycle.

(1) Background: Vaccinium vitis-idaea is a nutritionally and economically valuable natural wild plant species that produces berries useful for treating various diseases. There is growing interest in lingonberry, but there is limited information regarding lingonberry reference genes suitable for gene expression analyses of different tissues under various abiotic stress conditions. The objective of this study was to identify stable reference genes suitable for different lingonberry tissues in response to abiotic stress. (2) Methods: The delta Ct method and the GeNorm v3.5 and NormFinder v20 programs were used to comprehensively analyze gene expression stability. (3) Results: Actin Unigene23839 was the best reference gene for analyzing different cultivars, whereas Actin CL5740.Contig2 was the most suitable reference gene for analyzing different tissues and alkali stress. In contrast, 18S rRNA CL5051.Contig1 was the most stable reference gene under drought conditions. (4) Conclusions: These suitable reference genes may be used in future qRT-PCR analyses of different lingonberry tissues and the effects of abiotic stresses. Furthermore, the study data may be useful for functional genomics studies and the molecular breeding of lingonberry. In summary, internal reference genes or internal reference gene combinations should be carefully selected according to the experimental conditions to ensure that the generated gene expression data are accurate.

Selection and Validation of Reference Genes for qRT-PCR Analysis of Gene Expression in Tropaeolum majus (Nasturtium)

Tropaeolum majus (nasturtium) is an important ornamental and medicinal plant due to its colorful flowers, shield-shaped leaves, and richness in mineral elements and bioactive compounds. However, the key genes related to these important biological traits, as well as their expression patterns and functions, remain obscure. In this study, to choose appropriate reference genes for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, we screened 14 candidate genes from the transcriptome of T. majus and evaluated their expression stability. Through evaluation with four commonly used algorithms (geNorm, NormFinder, BestKeeper, and RefFinder), EXP1, EXP2, and TUB6 were found to be the most stably expressed genes among different organs, while EXP1 combined with CYP2 was identified as the optimal reference gene combination for seeds at different development stages. For all the tested samples, EXP1, EXP2, CYP2, and ACT2 were the most suitable reference genes. Moreover, the target gene KCS11 involved in very-long-chain fatty acid biosynthesis was employed to confirm the most and least stable reference genes in different organs, seeds at different development stages, and all the tested samples. The expression profiles of KCS11 were similar, with minor differences based on the analysis of different stable reference genes (either alone or in combination), while the expression profiles were diverse and the relative expression level was overestimated when using the least stable ones. These results suggest that the appropriate selection of reference genes is critical for the normalization of gene expression. Furthermore, the reference genes screened in this study will greatly improve the accuracy of the qRT-PCR quantification of candidate genes involved in the many biological characteristics of nasturtium.

Selection and validation of optimal reference genes for RT-qPCR analyses in Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae).

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza.

Development of reference genes for RT-qPCR analysis of gene expression in Pleurotus pulmonarius for biotechnological applications

Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics.

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in Lindera megaphylla.

Lindera megaphylla, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in L. megaphylla. In this study, 14 candidate genes were selected from the transcriptome database of L. megaphylla for RT-qPCR assay under different conditions. Results showed that helicase-15 and UBC28 were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was ACT7 and UBC36. UBC36 and TCTP were the best under cold treatment, while PAB2 and CYP20-2 were the best under heat treatment. Finally, a RT-qPCR assay of LmNAC83 and LmERF60 genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in L. megaphylla and will provide an important foundation for future genetic studies of this species.

Experimental assessment of robust reference genes for qRT-PCR in lung cancer studies.

Stable internal reference genes are crucial for quantitative real-time PCR (qRT-PCR) analyses in lung cancer studies. Widely used reference genes are mostly chosen by intuition or from pan-cancer transcriptome data and lack experimental validation by qRT-PCR in the context of lung cancer. This study evaluated the stability of candidate reference genes in lung cancer cell lines under normal homeostasis, hypoxia, and serum deprivation to screen for robust reference genes for qRT-PCR in lung cancer studies. The stability of reference gene combinations was also assessed. We found that most of the stably expressed genes from pan-cancer transcriptome analyses were not sufficiently stable under some of the tested conditions. CIAO1, CNOT4, and SNW1 were found to be the most stable reference genes under various conditions. Greater stability was achieved by combining more reference genes. We further used the hypoxia biomarker hypoxia-inducible factor (HIF)-2α to demonstrate that choosing inappropriate reference genes can lead to incorrect qRT-PCR results. We also found that the stable reference genes were irrelevant to malignancy, which may explain their stability under various conditions that cancer cells often encounter. This study provides a list of validated and stable qRT-PCR reference genes and reference gene combinations for lung cancer that may standardize qRT-PCR experiments in future lung cancer studies.

Reference genes selection for qRT-PCR analysis in various flowering transition events of Magnolia ×soulangeana ‘Changchun’

The appropriate reference genes are the prerequisite for gene expression analysis by qPCR, and their stability can affect the delicate and reliable results directly. Magnolia ×soulangeana ‘Changchun’ is a representative ornamental Magnolia that possesses a complex annual flowering cycle, including floral transition, dormancy, and twice blooming, and it is of great value and significance in investigating the flowering regulation of magnolias. In order to screen the appropriate reference genes related to various flowering transition events in ‘Changchun’, nine candidate reference genes were selected for qPCR analysis based on transcriptome data. Here, the expression stability of these nine genes in 63 different ‘Changchun’ samples was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). The results showed that GBP, UBQ, UBC and GAPDH were identified as the most stable gene in the floral transition, summer flowering initiation, spring flowering initiation and dormancy, respectively. Notably, UBQ showed the highest expression stability in the integrative flowering events set while UBC was suitable for the all samples set. Particularly, GBP was the relatively stable one in all sets. Further gene expression verification indicated that qPCR results using UBC+GBP as the reference genes were the most highly correlated with high throughput sequencing data. In general, the present study suggests that reference genes selection is developmental stage-dependent, and UBC+GBP is recommended as the best reference gene combination applied in qPCR assays of the integrative flowering events in ‘Changchun’. These findings will provide technical support for further molecular regulation studies of flowering events or other biological processes in magnolias, and even can be extended to other species.

Screening and validating of endogenous reference genes in Chlorella sp. TLD 6B under abiotic stress

Chlorella sp. TLD 6B, a microalgae growing in the Taklamakan Desert, Xinjiang of China, is a good model material for studying the physiological and environmental adaptation mechanisms of plants in their arid habitats, as its adaptation to the harsh desert environment has led to its strong resistance. However, when using real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the gene expression of this algae under abiotic stress, it is essential to find the suitable endogenous reference genes so to obtain reliable results. This study assessed the expression stability of 9 endogenous reference genes of Chlorella sp. TLD 6B under four abiotic stresses (drought, salt, cold and heat). These genes were selected based on the analysis results calculated by the three algorithmic procedures of geNorm, NormFinder, and BestKeeper, which were ranked by refinder. Our research showed that 18S and GTP under drought stress, 18S and IDH under salt stress, CYP and 18S under cold stress, GTP and IDH under heat stress were the most stable endogenous reference genes. Moreover, UBC and 18S were the most suitable endogenous reference gene combinations for all samples. In contrast, GAPDH and α-TUB were the two least stable endogenous reference genes in all experimental samples. Additionally, the selected genes have been verified to be durable and reliable by detecting POD and PXG3 genes using above endogenous reference genes. The identification of reliable endogenous reference genes guarantees more accurate RT-qPCR quantification for Chlorella sp. TLD 6B, facilitating functional genomics studies of deserts Chlorella as well as the mining of resistance genes.

Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components. Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression. In this study, eight candidate reference genes were selected from our previously published RNA-seq data, and primers for each candidate reference gene were designed and evaluated. The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples, including 12 subgroups of developing or abiotic-stressed tissues. Different combinations of stable reference genes were screened according to a comprehensive rank, which was synthesized from the results of four algorithms, including the geNorm, NormFinder, BestKeeper and ΔCt methods. The screened reference genes were verified by normalizing EjLGA1 in each subgroup. The obtained suitable combinations of reference genes for accurate normalization were GAPDH, EF1α and ACT for floral development; GAPDH, UBCE and ACT for fruit setting; EF1α, GAPDH and eIF2B for fruit ripening; ACT, EF1α and UBCE for leaves under heat stress; eIF2B, UBCE and EF1α for leaves under freezing stress; EF1α, TUA and UBCE for leaves under salt stress; ACT, EF1α and eIF2B for immature pulp under freezing stress; ACT, UBCE and eIF2B for immature seeds under freezing stress; EF1α, eIF2B and UBCE for both immature pulp and seeds under freezing stress; UBCE, TUB and TUA for red-fleshed fruits under cold-storage stress; eIF2B, RPS3 and TUB for white-fleshed fruits under cold-storage stress; and eIF2B, UBCE and RPS3 for both red- and white-fleshed fruits under cold-storage stress. This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat. To our knowledge, this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E. japonica. The use of the three most stable reference genes could increase the reliability of future quantification experiments.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.

Identification of optimal reference genes for gene expression normalization in human osteosarcoma cell lines under proliferative conditions.

The molecular pathogenesis and therapeutic target research studies on osteosarcoma (OS) have developed well during the last few years using various OS cell lines with reverse transcription quantitative polymerase chain reaction (RT-qPCR). However, the identification of suitable reference genes of RT-qPCR for OS cell lines has not been reported. Here, we conducted the normalization research of 12 reference genes (GAPDH, ACTB, 18S, B2M, ALAS1, GUSB, HPRT1, HMBS, PPIA, PUM1, RPL29, and TBP) for gene expression analysis in four kinds of human OS cell lines (U2OS, Saos-2, HOS, and MG-63) to improve the investigation of molecular mechanisms and the accuracy of diagnosis and prognostic molecular targets of OS. The gene expression stability and applicability of the 12 reference gene candidates were determined using geNorm, NormFinder, and BestKeeper software. The results indicated that PUM1 and the combination of PPIA + ALAS1 were recommended as the optimal reference gene in these four different sources of human OS cell lines under proliferative conditions. The present study identified the most suitable reference genes and reference gene combinations for OS cell lines under proliferative conditions in order to use in gene expression profile analysis. A reliable standardized method has the potential to improve the understanding of the biological mechanisms underlying OS in the future.

Reference genes selection for expression studies in Maconellicoccus hirsutus (Green) (Pseudococcidae: Hemiptera) under specific experimental conditions.

Maconellicoccus hirsutus is a destructive pest which causes severe losses of agricultural and horticultural crops. For the management of M. hirsutus, many insecticides have been used and it has been exposed to insufficient dosage or uneven spray coverage which resulted in the development of insecticide resistance. Xenobiotic metabolism can be better understood with the help of gene expression studies by unveiling the underlying molecular mechanisms. The qRT-PCR is the simplest method to analyse gene expression, however, it highly relies on suitable reference genes concerning the different experimental conditions. We evaluated the stability of five reference genes in two sets of experimental conditions viz. developmental stages (nymphs and adults) and agrochemical stress (GA3 and Buprofezin sprayed) against M. hirsutus, using different softwares-NormFinder, geNorm, BestKeeper, and RefFinder. The study revealed that ATP51a and GAPDH can be used as reference genes for gene expression studies when exposed to Gibberellic acid. Additionally, the study revealed that the ideal pair of reference genes for data validation in M. hirsutus treated with Buprofezin was GAPDH and β-tubulin. The ideal reference gene combination for various developmental stages was found to be 28S and Actin. According to the study, GAPDH can be utilized as a reliable reference gene in the agrochemical (GA3 and Buprofezin) exposure set. The genes can be utilized as a suitable reference for qRT-PCR gene expression studies of xenobiotic metabolism to understand the underlying molecular mechanism, which will help further to design suitable management strategies.

Identification and Validation of Common Reference Genes for Normalization of Esophageal Squamous Cell Carcinoma Gene Expression Profiles.

Esophageal squamous cell carcinoma (ESCC) is one of the subtypes of esophageal cancer with Chinese characteristics, and its five-year survival rate is less than 20%. Early diagnosis is beneficial to improving the survival rate of ESCC significantly. Quantitative Real-Time Polymerase Chain Reaction is a high-throughput technique that can quantify tumor-related genes for early diagnosis. Its accuracy largely depends on the stability of the reference gene. There is no systematic scientific basis to demonstrate which reference gene expression is stable in ESCC and no consensus on the selection of internal reference. Therefore, this research used four software programs (The comparative delta-Ct method, GeNorm, NormFinder, and BestKeeper) to evaluate the expression stability of eight candidate reference genes commonly used in other tumor tissues and generated a comprehensive analysis by RefFinder. Randomly selected transcriptome sequencing analysis confirmed the SPP1 gene is closely related to ESCC. It was found that the expression trend of SPP1 obtained by RPS18 and PPIA as internal reference genes were the same as that of sequencing. The results show that RPS18 and PPIA are stable reference genes, and PPIA + RPS18 are a suitable reference gene combination. This is a reference gene report that combines transcriptome sequencing analysis and only focuses on ESCC, which makes the quantification more precise, systematic, and standardized, and promotes gene regulation research and the early diagnosis of ESCC in the future.