Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Effects of hypoxia on mRNA expression of housekeeping genes in rat brain tissue and primary cultured neural cells

Internal standards are critical for quantitative RNA analyses.Housekeeping genes are often used as internal standards with the assumption that their express-ion levels remain relatively constant in different experi-mental conditions.In this study,four commonly used housekeeping genes,Glyceraldehyde-3-phosphate dehy-drogenase(GAPDH),β-actin,28S rRNA and 18S rRNA were selected to test whether this assumption is tenable under hypoxic conditions.We tested the RNA expression level of these four genes in different hypoxic conditions.Rats subjected to acute hypoxia for 2 hours were used for tissue detection.Primary cultured neural stem cells from E13 fetal rats were treated with 3%O_(2) or 10%O_(2) for 24 hours for in vitro experiments.In both experiments,expression levels of 28S rRNA and 18S rRNA were constant,independent of hypoxia types.However,expression levels of GAPDH and b-actin were all changed in all kinds of hypoxic conditions.In particu-lar,the mRNA expression level of GAPDH was increased by 43.4%under 3%O_(2) hypoxic conditions.These results suggest that 18S rRNA and 28S rRNA are reliable internal controls for comparative analyses of transcrip-tion under hypoxia.GAPDH appears particularly unfa-vorable for this purpose in hypoxic conditions.

Identification of Rhizobia Isolated from Nodules of Mexican Commercial Soybean Varieties

Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybean varieties in high-production regions. Multilocus sequence analysis (MLSA) was employed for enhanced species resolution. The study identified six Bradyrhizobium species: Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 6, Bradyrhizobium elkanii USDA 76, Bradyrhizobium neotropicale, Bradyrhizobium lablabi, and Bradyrhizobium icense. Bradyrhizobium japonicum USDA 110 predominated in the soils, displaying symbiotic preference for the Huasteca 400 variety. However, phylogenetic analysis didn't reveal a clear association between strains, soil, and soybean variety. This research sheds light on the diversity of rhizobia in Mexican soybean cultivation, contributing to the understanding of symbiotic relationships in soybean production systems.

Exploring valid internal-control genes in Porphyra yezoensis(Bangiaceae) during stress response conditions

To screen the stable expression genes related to the stress （strong light, dehydration and temperature shock） we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y（II） and Fv/Fm were significantly affected when stress was imposed on the thalli of Porphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress （at salinity 90） on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-la showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill)stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.

MLST analysis of genetic diversity of Bacillus coagulans strains to evaluate effects on constipation model

Bacillus coagulans can help ameliorate or prevent gastrointestinal diseases, but the genetic relationships among B. coagulans isolates are not well studied. Multilocus sequence typing analysis was conducted on 57 isolates of B. coagulans from 22 provinces or autonomous regions in China. B. coagulans isolates were highly diverse and a total of 33(sequence typings) STs were found. These isolates had a weak clonal population structure and strong indications of intraspecies recombination. The evolution direction of B. coagulans was not correlated with geography or isolation source. Fifteen strains were selected for further analysis based on proximity relationships from the phylogenetic tree. Five isolates(B. coagulans-1, B. coagulans-10, B. coagulans-39, B. coagulans-70 and B. coagulans-71) with good spore-forming ability relative to the rest of the isolates were evaluated for constipation relief. B. coagulans-39 significantly relieved constipation symptoms in mice by regulating intestinal flora, increasing the production of short-chain fatty acids and restoring the level of gastrointestinal regulatory peptides. Comparative genomic analysis showed the beneficial effects of B. coagulans-39 might be associated with specific functional genes that are involved in the utilization of various carbohydrates as primary substrates and short-chain fatty acid production.

Comprehensive Analysis of Ubiquitously Expressed Genes in Humans from A Data-driven Perspective

Comprehensive characterization of spatial and temporal gene expression patterns in humans is critical for uncovering the regulatory codes of the human genome and understanding the molecular mechanisms of human diseases.Ubiquitously expressed genes(UEGs)refer to the genes expressed across a majority of,if not all,phenotypic and physiological conditions of an organism.It is known that many human genes are broadly expressed across tissues.However,most previous UEG studies have only focused on providing a list of UEGs without capturing their global expression patterns,thus limiting the potential use of UEG information.In this study,we proposed a novel data-driven framework to leverage the extensive collection of40,000 human transcriptomes to derive a list of UEGs and their corresponding global expression patterns,which offers a valuable resource to further characterize human transcriptome.Our results suggest that about half(12,234;49.01%)of the human genes are expressed in at least 80%of human transcriptomes,and the median size of the human transcriptome is 16,342 genes(65.44%).Through gene clustering,we identified a set of UEGs,named LoVarUEGs,which have stable expression across human transcriptomes and can be used as internal reference genes for expression measurement.To further demonstrate the usefulness of this resource,we evaluated the global expression patterns for 16 previously predicted disallowed genes in islet beta cells and found that seven of these genes showed relatively more varied expression patterns,suggesting that the repression of these genes may not be unique to islet beta cells.

Rice Expression Database(RED)：An integrated RNA-Seq-derived gene expression database for rice

Rice is one of the most important stable food as well as a monocotyledonous model organism for the plant research community.Here,we present RED（Rice Expression Database;http：//expression.ic4r.org）,an integrated database of rice gene expression profiles derived entirely from RNA-Seq data.RED features a comprehensive collection of 284 high-quality RNA-Seq experiments,integrates a large number of gene expression profiles and covers a wide range of rice growth stages as well as various treatments.Based on massive expression profiles,RED provides a list of housekeeping and tissue-specific genes and dynamically constructs co-expression networks for gene（s） of interest.Besides,it provides user-friendly web interfaces for querying,browsing and visualizing expression profiles of concerned genes.Together,as a core resource in BIG Data Center,RED bears great utility for characterizing the function of rice genes and better understanding important biological processes and mechanisms underlying complex agronomic traits in rice.

The Function of Surfeit 4 Gene in vivo

Surfeit 4(Surf 4), one of the identified housekeeping genes, was previously thought to encode a transmembrane protein that acts as a cargo receptor to maintain basic cellular functions. In recent years, Surf 4 gene is overexpressed in some types of cancer, involved in the exosome signaling pathway in gastrointestinal stromal tumors, and played the role of an oncogene in ovarian cancer stem cells. In this paper,we review the characteristics and protein localization of Surf 4 gene, as well as its possible physiological functions and pathogenesis in various diseases and cancers,expecting to discuss its clinical application value.

Suppressing tawny crazy ant (Nylanderia fulva) by RNAi technology

A bstract The tawny crazy ant(Nylanderia fulva)is a new invasive pest in the United States.At present,its management mainly relies on the use of synthetic insecticides,which are generally ineffective at producing lasting control of the pest,necessitating alternative environmentally friendly measures.In this study,we evaluated the feasibility of gene silencing to control this ant species.Six housekeeping genes encoding actin(NfActin),coatomer subunit β (NfCOPP),arginine kinase(NfArgK),and V-type proton ATPase subunits A(NfvATPaseA),B(NfvATPaseB)and E(NfvATPaseE)were cloned.Phylogenetic analysis revealed high sequence similarity to homologs from other ant species,particularly the Florida carpenter ant(Camponotus floridanus).To silence these genes,vector L4440 was used to generate six specific RNAi constructs for bacterial expression.Heat-inactivated,dsRNA-expressing Escherichia coli were incorporated into artificial diet.Worker ants exhibited reduced endogenous gene expression after feeding on such diet for 9 d.However,only ingestion of dsRNAs of NfCOPfi(a gene involved in protein trafficking)and NfArgK(a cellular energy reserve regulatory gene in invertebrates)caused modest but significantly higher ant mortality than the control.These results suggest that bacterially expressed dsRNA can be orally delivered to ant cells as a mean to target its vulnerabilities.Improved efficacy is necessary for the RNAi-based approach to be useful in tawny crazy ant management.

Effects of Long-Term Fertilization Strategies on Soil Productivity and Soybean Rhizobial Diversity in a Chinese Mollisol

Rhizobial diversity is affected by interactions between soil features, fertilization strategy, and cropping system. However, interactions among the rhizobial community, chemical-organic manure fertilization, and plant production have not been well documented in Mollisols from long-term experiments. Aimed at maintaining and recovering the productivity of Chinese Mollisols, a long-term fertilization experiment had been carried out for 29 years under a wheat-maize-soybean rotation system, involving the application of recycled organic manure (ROM), chemical fertilizers (N, P, and/or K), or ROM plus N, P, and/or K. In the present study, the effects of different treatments were evaluated by determining soil physicochemical features, soybean production, and soybean rhizobial diversity. The results showed that application of ROM plus NPK maintained or increased soil fertility, which was accompanied by higher production and higher diversity of rhizobia, as compared with the other treatments. The negative association of Bradyrhizobium japonicum with N fertilizer, positive association of B. diazoefficiens with soil pH, and alleviation of N-inhibition on the diversity of Bradyrhizobium by the addition of ROM were recorded as new findings. Therefore, application of ROM or ROM plus NPK could be a feasible strategy for maintaining and recovering the fertility of Chinese Mollisols, whereas rhizobial diversity could be an indicator of soil fertility.