Candidate Reference Genes Research Articles

Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress.

The principal objective of this study was to screen and verify reference genes appropriate for gene expression evaluation during plant growth and development under distinct growth conditions. Nine candidate reference genes were screened based on garlic transcriptome sequence data. RT-qPCR was used to detect the expression levels of the aforementioned reference genes in specific tissues under drought and cold stress. Then, geNorm, NormFinder, BestKeeper, and ReFinder were used to consider the consistency of the expression levels of candidate reference genes. Finally, the stress-responsive gene expression of ascorbate peroxidase (APX) was quantitatively evaluated to confirm the chosen reference genes. Our results indicated that there were variations in the abundance and stability of nine reference gene transcripts underneath cold and drought stress, among which ACT and UBC-E2 had the highest transcript abundance, and 18S rRNA and HIS3 had the lowest transcript abundance. UBC and UBC-E2 were the most stably expressed genes throughout all samples; UBC and UBC-E2 were the most stably expressed genes during cold stress, and ACT and UBC were the most stably expressed genes under drought stress. The most stably expressed genes in roots, pseudostems, leaves, and cloves were EF1, ACT, HIS3, UBC, and UBC-E2, respectively, while GAPDH was the most unstable gene during drought and cold stress conditions and in exclusive tissues. Taking the steady reference genes UBC-E2, UBC, and ACT as references during drought and cold stress, the reliability of the expression levels was further demonstrated by detecting the expression of AsAPX. Our work thereby offers a theoretical reference for the evaluation of gene expression in garlic in various tissues and under stress conditions.

Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary.

Normalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed. Six candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively. Our findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

Validation of reference genes for quantitative real‐time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

AbstractIn quantitative real‐time polymerase chain reaction (qRT‐PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up‐ or down‐regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT‐PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT‐PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.

Effect of Savirin or Ticagrelor Treatment on the Expression of Commonly Used Reference Genes in Staphylococcus aureus

Reference genes are frequently used for the normalization of quantitative reverse transcriptase PCR (qRTPCR) data in gene expression studies. Staphylococcus aureus is one of the most common causes of biofilm-related infections. Savirin and ticagrelor show in vitro as well as in vivo antibiofilm activity against S. aureus. The main aim of this study was to identify the most stably expressed reference genes to study the effect of these molecules on genes in a strong biofilm producing S. aureus isolate isolated from biofilm-related infection. Quantitative real-time PCR was performed by using relative quantification method. Four different algorithms, delta Ct, normfinder, bestkeeper, and genorm, followed by a comprehensive analysis was used to identify the most stable reference genes from a list of sixteen different candidate reference genes. All four algorithms reported different results, with some comparable findings among some methods. In the comprehensive analysis of the results of all the algorithms used, the most stable reference genes found were spa, rpoD, and pyk for savirin treatment experiment and gapdH, gyrA, and gmk for ticagrelor treatment experiment. The optimal number of reference genes required was two for both the experimental conditions. Despite having some drawbacks, each algorithm can reliably determine an appropriate reference gene independently. However, based on consensus ranking and the required optimal number of reference genes reported, spa and rpoD were the most appropriate reference genes for savirin treatment experiment, and gapdH and gyrA were most appropriate for ticagrelor treatment experiment. This study provides baseline data on reference genes to study the effect of savirin or ticagrelor treatment on the expression of potential reference genes in S. aureus. We recommend prior re-validation of reference genes on a case-by-case basis before they can be used.

Identification of reference genes for RT-qPCR analysis across kiwifruit species under iron deficiency conditions

Selecting appropriate reference genes is crucial for the accuracy of gene expression analysis. Although rootstock species play vital roles in kiwifruit production, the reference genes has not been identified in kiwifruit roots and across kiwifruit species. Additionally, iron (Fe) deficiency has been frequently observed in kiwifruit orchards, but the reference genes is not validated. Here, we assessed ten candidate reference genes: actin (ACT), actin-depolymerizing factor family protein (ADF), ADP-ribosylation factor (ARF), eukaryotic translation initiation factor 5A (eIF5A), ribosomal protein L19 (RPL19), 40S ribosomal protein S17 (RPS17), suppressor of G2 allele of SKP1 (SGT1), translationally-controlled tumor protein (TCTP), translation initiation factor 2-γ (TIF2-γ) and ubiquitin-conjugating enzyme E2 variant (UBE2V), for their suitability as reliable internal controls in roots, stems and leaves of four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) under direct and bicarbonate-induced Fe deficiency conditions. The expression stability of the candidate genes was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Based on the comprehensive ranking, we recommended TCTP + UBE2V as the best reference genes for roots, UBE2V + ACT for stems, RPL19 + ARF for leaves, and UBE2V + RPL19 for all the tissues. The reliability of the recommended reference genes was verified by six target genes involved in Fe uptake and transport. These results provide a basis for improving the accuracy of gene expression analysis and elucidating the molecular mechanisms of Fe deficiency adaptation in kiwifruit species.

Identification of reliable reference genes for gene expression studies in mouse models under microplastics stress

Microplastics (MPs) and nanoplastics (NPs) have been deemed to be newly emerged contaminants interfering with various physiological processes closely related with gene expression alteration. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) serves as a powerful tool to assess gene expression, however highly dependent on a reliable reference gene. Therefore, it is necessary to identify stable reference genes for gene expression study under MP or NP stress. We constructed a mouse model postexposure to polypropylene microplastics (PP-MPs) to assess PP-MPs bioaccumulation in kidney, evaluate the kidney pathological changes, and then explore potential reference genes via RT-qPCR. Although the hematoxylin-eosin staining showed no obvious damage in kidney tissues, we observed significant PP-MPs accumulation in kidney using Raman spectra analysis supported by spectral multivariate analysis. The expression of 19 candidate reference genes were examined, including the commonly used ones of β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Cytochrome c oxidase subunit 4I1 (Cox4i), Histocompatibility 13 (H13) and ribosomal protein. Their expression stability and reliability were assessed by the combination of four algorithms including geNorm, NormFinder, BestKeeper and Delta Cq. The geNorm analysis revealed that the top three genes with the lowest variability were Cox4il, Rps9 and Gapdh, whereas NormFinder results ranked Rps3, Cox4il and Rps18 as the top three ones. Rpl15, Cox4i1 and Rps3 were the most reliable reference genes in BestKeeper results, and Delta Cq proposed Rps3 and Cox4il as the stable genes. The overall ranking indicated by GMR value gave the five most stable reference genes (Cox4i1, Rps3, Rps9, Rps18 and Gapdh). Three genes associated with different biochemical processes (Atp5f1, Crebbp and Dele1) were chosen to verify the characterized reference genes using the least stable gene as a control, exhibiting different expression profiles and implying the essentiality to select the reliable reference genes. Our results documented the expression fluctuations of acknowledged reference gene (Ubc) and proposed a set of reliable reference genes for future studies of gene expression profiles in MP treated mouse models.

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 and validation of reference genes for quantitative real-time polymerase chain reaction analyses of Serratia ureilytica DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.

Reference gene selection for normalizing gene expression using quantitative real‐time PCR in Haemaphysalis longicornis

AbstractThe Asian longhorned tick, Haemaphysalis longicornis, the dominant species of Ixodidae in Korea, has a wide distribution in East Asia, far‐East Russia, and Western Pacific countries, and has recently been discovered in the Eastern states of the United States of America. H. longicornis transmits various pathogens, including Babesia ovate, Rickettsia japonica, and severe fever with thrombocytopenia syndrome virus (SFTSV). Considering its medical importance, in order to understand the physiology of H. longicornis, it is crucial to determine the expression of the genes of interest. Although quantitative real‐time PCR (qRT‐PCR) has been widely used to analyze gene expression, stably‐expressed internal reference genes across samples of different conditions should be selected for the accurate normalization of target gene expression levels. Therefore, in this study, we investigated the expression levels of five candidate reference genes, namely ACT, RPP0, RPL23, TUB, and GAPDH, in H. longicornis under different conditions, including different collection months, developmental stages, and SFTSV infection status. Using four software programs, namely, NormFinder, BestKeeper, geNorm, and RefFinder, their expression stabilities were evaluated. Subsequently, a single gene between RPL23 and RPP0 was validated, which was found to be most stable reference gene after comparing the expression levels of HSP70 determined using different normalization methods.

Identification of RT-qPCR reference genes suitable for gene function studies in the pitaya canker disease pathogen Neoscytalidium dimidiatum

Neoscytalidium dimidiatum is the main causal agent of pitaya canker. Most studies of virulence and pathogenicity genes have measured expression levels using real-time quantitative polymerase chain reaction (RT-qPCR). Suitable reference genes are essential for ensuring that estimates of gene expression levels by RT-qPCR are accurate. However, no reference genes can be robustly applied across all contexts and species. No studies to date have evaluated the most effective reference genes for normalizing gene expression levels estimated by RT-qPCR in N. dimidiatum. In this study, RT-qPCR data for individual candidate reference genes were analyzed using four different methods: the delta Ct method and the geNorm, NormFinder, and BestKeeper algorithms. We evaluated the utility of eight candidate reference genes (18S rRNA, Actin (1), Actin (2), Actin, GAPDH (1), GAPDH (2), UBQ, and Tubulin) for normalizing expression levels estimated by RT-qPCR in N. dimidiatum at different developmental stages, at different temperatures, and during interaction with pitaya. All candidate reference genes were suitable for gene expression analysis except for Actin (2). Tubulin and Actin (1) were the most stably expressed reference genes under different temperatures. Actin (1) and Actin were the most stably expressed reference genes in N. dimidiatum at different developmental stages. Tubulin and UBQ were the most stably expressed reference genes during interaction with pitaya. Actin and 18s rRNA were the most stably expressed across all experimental conditions. Subsequently, Tubulin and UBQ were further investigated in analyses of pectinase expression during the pitaya–N. dimidiatum interaction. Our results provide insights that will aid future RT-qPCR studies of gene expression in N. dimidiatum.

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

BackgroundNitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT–qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT–qPCR analysis.ResultsIn this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions.ConclusionThis study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Selection of Reference Genes for Transcription Studies Considering Co-Regulation and Average Transcriptional Stability: Case Study on Adventitious Root Induction in Olive (Olea europaea L.) Microshoots

Selection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the between-sample transcriptionally more stable genes, and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried out through generally accepted strategies, each having different strengths and weaknesses. The present study proposes reinforcement of the normalization of gene expression data by integrating analytical revision at critical steps of those accepted procedures. In vitro olive adventitious rooting was used as an experimental system. Candidate RGs were ranked according to transcriptional stability according to several methods. An algorithm of one of these programs (GeNorm) was adapted to allow for partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to choose the more appropriate set of RGs, the achieved results were analytically revised, with special emphasis on biasing effects such as co-regulation. The obtained putative RG sets were also tested for cases restricted to fewer variables. The set formed by the genes H2B, OUB and ACT is valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays. Such internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.

Reference genes identification for qRT-PCR normalization of gene expression analysis in Cucumis sativus under Meloidogyne incognita infection and Pseudomonas treatment.

qRT-PCR is a common and key technical means to study gene expression in biological research. However, reliability and accuracy of quantification by qRT-PCR is entirely dependent on the identification of appropriate reference genes. Cucumber as an economical vegetable is widely cultivated worldwide and is subject to serious nematode infection, especially from M. incognita. Plant could employ beneficial soil bacteria in the rhizosphere to enhance plant adaptability to various stresses. In this study, the optimal reference genes in cucumber under M. incognita stress and Pseudomonas treatment were calculated and confirmed. A total of thirteen candidate reference genes were identified across three different treatments. Of these, geNorm, NormFinder and BestKeeper programs combined RefFinder software identified EF1 and UBI are the most suitable reference gene in the root knot and whole root of cucumber infected M. incognita, respectively, and CACS is the most suitable reference gene in the whole root of cucumber treated by Pseudomonas. The work first validated the most suitable reference genes for the normalization gene expression in cucumber by nematode infected or Pseudomonas inoculated, and these results would facilitate the further research on M. incognita or Pseudomonas soil rhizosphere microbe interaction with cucumber.

Selection and Evaluation of Reference Genes for Quantitative Real-Time PCR in Tomato (Solanum lycopersicum L.) Inoculated with Oidium neolycopersici

In order to screen out the most stable reference genes in tomatoes under powdery mildew (PM) stress and study the expression of related genes in the interaction between tomato and PM more accurately, this study will provide a calibration basis for the expression of related functional genes. In this study, the expression stabilities of eight tomato candidate reference genes of EF1α, L33, Act, Ubi, GAPDH, UK, CAC and TIP41 in susceptible tomato and resistant tomatoes under PM stress were ranked using four different computation programs, including geNorm, Normfinder, BestKeeper and the comparative ∆CT method. Then RefFinder was used to analyze the ranking results of four kinds of software comprehensively. Finally, the selected reference genes were validated by the target gene SlMLO1. The results of geNorm showed that the normalization of qRT-PCR using two reference genes could meet the requirements. The comprehensive analysis of RefFinder showed that the most stable reference genes were Act and EF1α for both tomato varieties. The combination of Act and GAPDH was most stable in susceptible tomato ‘MM’. The combination of Act and EF1α was most stable in resistant tomato ‘62579′. Generally, the Act was the most stable reference gene in the two tomato varieties under PM stress. This study will lay a foundation for the related functional gene expression research in tomatoes under PM 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.

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) strongly depends on the stability of reference gene. It is essential to select a suitable reference gene in order to obtain reliable RT-qPCR results when gene expression profiles were evaluated. Callerya speciosa, a traditional Chinese medicine, has a long cultivation history in south China. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L34), ACTIN (actin), TUA2 (tubulin alpha chain), TUB1 (tubulin beta-1 chain), TIF5 (eukaryotic translation initiation factor 5A), UBQ (polyubiquitin), and EF2 (elongation factor 2), were selected from the transcriptome databases, and their expression stability under six different experimental conditions (developmental stages, tissues, MeJA treatment, GA3 treatment, CPPU treatment, and PP333 treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the best stability under most of the hormone treatments. TUA2 and EF2 were the two least suitable ones as reference genes in C. speciosa. The expression pattern of CsMYB36, a gene associated with the regulation of isoflavonoid biosynthesis in C. speciosa, verified the accuracy of our experimental results. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa and other Callerya species in the future.

Identification of RNU44 as an Endogenous Reference Gene for Normalizing Cell-Free RNA in Tuberculosis.

Normalization of cell-free RNA (cf-RNA) concentration can be affected by variable experimental conditions and thus impact the performance of their diagnostic potential. Our study aimed to identify appropriate endogenous reference genes for cf-RNA biomarker evaluation in the diagnosis of tuberculosis (TB). Subjects consisting of patients with TB with and without malignancy, patients with pneumonia, and healthy controls were recruited. Candidate reference genes were screened and identified by literature reviewing and RNA-Seq analysis. Expression levels of the candidate genes were determined by reverse-transcription real-time quantitative polymerase chain reaction in plasma from patients with TB and healthy controls. The stability of gene expression was assessed by geNorm, NormFinder, BestKeeper, the Comparative Delta Ct method, and RefFinder. Differential expression of 2 small RNAs (sRNAs) encoding by genome of Mycobacterium tuberculosis in plasma of patients with TB were determined by both absolute quantification and relative quantification with candidate reference genes. According to the stability ranking analyzed with the 5 computational programs, the top 4 candidates-miR-93, RNU44, miR-16, and glyceraldehyde 3-phosphate dehydrogenase-were used to normalize the transcript levels of 2 mycobacterial sRNAs, MTS2823 and MTS1338, which were observed to have higher copy numbers in the plasma of patients with TB. Normalization with RNU44 displayed significantly higher levels of the 2 M tuberculosis sRNAs in the patients' plasma than those of healthy controls. RNU44 was demonstrated as a proper endogenous gene for cf-RNA normalization in TB diagnosis.

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool.

Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy-to-use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead-based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin-induced RNA transcripts. Using a 50-gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time-consuming reverse-transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead-based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130-142. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Tissues from Bumble Bees (Bombus Terrestris) of Different Lines.

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.