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.

Equalization Reconstruction Algorithm Based on Reference Signal Frequency Domain Block Joint for DTMB-Based Passive Radar

Channel equalization plays a pivotal role within the reconstruction phase of passive radar reference signals.In the context of reconstructing digital terrestrial multimedia broadcasting(DTMB)signals for low-slow-small(LSS)target detection,a novel frequency domain block joint equalization algorithm is presented in this article.From the DTMB signal frame structure and channel multipath transmission characteristics,this article adopts a unconventional approach where the delay and frame structure of each DTMB signal frame are reconfigured to create a circular convolution block,facilitating concurrent fast Fourier transform(FFT)calculations.Following equalization,an inverse fast Fourier transform(IFFT)-based joint output and subsequent data reordering are executed to finalize the equalization process for the DTMB signal.Simulation and measured data confirm that this algorithm outperforms conventional techniques by reducing signal errors rate and enhancing real-time processing.In passive radar LSS detection,it effectively suppresses multipath and noise through frequency domain equalization,reducing false alarms and improving the capabilities of weak target detection.

Defining the We-map Reference Frames and Providing Mathematical Expressions for Transformation Relations

We-map is an interactive mobile map that can be easily communicated and applied on personal electronic devices,such as personal computers and mobile phones.Therefore,the study of direction systems and coordinate systems is critical,and exploring reference frames is essential in direction and coordinate systems.Despite its significance,existing research on We-map lacks specific solutions for the exploration of reference frames is indispensable for the establishment of accurate direction and coordinate systems.In this paper,we endeavor to address this gap by elucidating the significance of We-map reference frames,defining them with mathematical constraints,summarizing their nature and characteristics,deriving their transformation relationships and representing them through mathematical formulars and equations.Our work contributes to the fundamental theory of We-map and provides valuable systems and support for the mathematical foundation of We-map,map production,and platform development.Ultimately,this research serves to advance the development of We-map.

Comparison study on measurement of rail weld joint between inertial reference method and multi-point chord reference method

Purpose – Straightness measurement of rail weld joint is of essential importance to railway maintenance. Dueto the lack of efficient measurement equipment, there has been limited in-depth research on rail weld joint with a5-m wavelength range, leaving a significant knowledge gap in this field.Design/methodology/approach – In this study, the authors used the well-established inertial referencemethod (IR-method), and the state-of-the-art multi-point chord reference method (MCR-method). Two methodshave been applied in different types of rail straightness measurement trollies, respectively. These instrumentswere tested in a high-speed rail section within a certain region of China. The test results were ultimatelyvalidated through using traditional straightedge and feeler gauge methods as reference data to evaluate the railweld joint straightness within the 5-m wavelength range.Findings – The research reveals that IR-method and MCR-method produce reasonably similar measurementresults for wavelengths below 1 m. However, MCR-method outperforms IR-method in terms of accuracy forwavelengths exceeding 3 m. Furthermore, it was observed that IR-method, while operating at a slower speed,carries the risk of derailing and is incapable of detecting rail weld joints and low joints within the track.Originality/value – The research compare two methods’ measurement effects in a longer wavelength rangeand demonstrate the superiority of MCR-method.

Reference Interval of Thyroidal Function of Women in the Early,Middle and Late Pregnancy in Jingzhou City,Hubei Province

[Objectives]To study the reference value of thyroid function in healthy women of childbearing age during different pregnancies(early,middle and late pregnancy)in Jingzhou City.[Methods]362 healthy women of childbearing age during different pregnancies(early,middle and late pregnancy)were selected as the study group,and 360 non-pregnant women were selected as the control group.Serum thyroid hormone levels in the two groups were detected,and the reference value was expressed as the median(M)and 95%confidence interval(CI).On this basis,the chart of changing trend of thyroid hormone in the early,middle and late pregnancy were plotted.[Results]Compared with the control group,FT3,TT4,TT3,FT4and TSH in the early,middle and late pregnancy were significantly different in the study group(P<0.05).In the study group,there were significant differences in the early,middle and late pregnancy(P<0.05).[Conclusions]This trend chart has certain reference value in the screening,diagnosis and treatment of thyroid diseases in healthy women of childbearing age during different pregnancies(early,middle and late pregnancy)in Jingzhou City of Hubei Province.

Review of Two Years of Surgical Activities of the General Surgery Department of the Reference Health Center of Commune I of Bamako Mali

The objectives of this work were to evaluate the surgical activities carried out in the general surgery department of the Reference Health Center of Commune I of Bamako, to describe the sociodemographic characteristics of the operated patients, to determine the main pathologies encountered and to evaluate qualitatively the result of the treatment. In order to improve performance, and the quality of care, and to identify common pathologies in the surgical department, we undertook a retrospective study on surgical activities from January 2009 to December 2010. At the end of this study, out of 474 men and 187 women (equal sex ratio 2.53);we were able to determine the frequency of surgical pathologies. Farmers, housewives and pupils/students were the most represented with 25.9% respectively;20% and 13.3%. The most frequently observed pathologies were wall hernia (44.8%), prostate adenoma (12%) and acute appendicitis (10.5%). The average length of hospitalization was 3.43 days. Infectious complications affected 25 patients (3.8% of cases) and a death rate of 0.45% (i.e. 3 patients). The average cost of care was 53,500 FCFA. Indeed, the reality of surgical practice in health centers was not the same because of the level of skills of practicing surgeons.

A Reference Vector-Assisted Many-Objective Optimization Algorithm with Adaptive Niche Dominance Relation

It is still a huge challenge for traditional Pareto-dominatedmany-objective optimization algorithms to solve manyobjective optimization problems because these algorithms hardly maintain the balance between convergence and diversity and can only find a group of solutions focused on a small area on the Pareto front,resulting in poor performance of those algorithms.For this reason,we propose a reference vector-assisted algorithmwith an adaptive niche dominance relation,for short MaOEA-AR.The new dominance relation forms a niche based on the angle between candidate solutions.By comparing these solutions,the solutionwith the best convergence is found to be the non-dominated solution to improve the selection pressure.In reproduction,a mutation strategy of k-bit crossover and hybrid mutation is used to generate high-quality offspring.On 23 test problems with up to 15-objective,we compared the proposed algorithm with five state-of-the-art algorithms.The experimental results verified that the proposed algorithm is competitive.

Pathological and Etiological Aspects of Nephrotic Syndrome at the Niamey General Reference Hospital

Introduction: Studies have been conducted on nephrotic syndrome in Niger. The study aimed to determine the histological and etiological aspects of nephrotic syndrome. Patients and Method: This was a retrospective study from February 1st, 2018 to January 31st, 2024. All patients with nephrotic syndrome who underwent renal biopsy were included. Samples were analyzed at the anatomy-cytology pathology laboratory of the Faculty of Medicine in Dakar (Senegal). The variables studied included clinical, biological, histological and etiological characteristics. Data were analyzed using Excel 2013 and Epi-info 7.2.0 software. Results: The study included 119 patients with nephrotic syndrome. Prevalence of nephrotic syndrome was 11.24%. The male-to-female ratio was 2.25:1. The mean age at diagnosis was between 34.5 ± 18.84 years. Edema was the reason for admission in 40.34% of cases. The nephrotic syndrome was impure in 63.86% of cases. Nine histological lesions were identified. Focal and segmental glomerulosclerosis (40.09%), minimal change disease (23.53%), membranous nephropathy (13.45%), diabetic nephropathy (10.92%), membranous proliferative glomerulonephritis (3.36%), acute glomerulonephritis (3.36%), glomerular thrombotic microangiopathy (2.52%), non-IgA mesengial proliferative glomerulonephritis (1.68%) and amyloidosis (0.84%). Nephrotic syndrome was primary in 57.98% of cases. Secondary etiologies were dominated by diabetes (11.76%), followed by hepatitis B virus (9.24%), lupus, lymphoma, malaria, syphilis, cryoglobulinemia, sickle cell disease and HIV. Conclusion: Future studies should investigate the causes of glomerulopathy secondary to chronic tubulointerstitial lesions.

一个传奇的品牌,一个经典的系列 KEF与TheReference系列的故事

回顾2023年,KEF的产品又再斩获众多不同定位、不同类型的各种Hi-Fi音响奖项。在更新MAT技术后,KEF全线新Meta系列在音质方面的提升是备受肯定的。其中最受瞩目的自然是KEF当家的Reference系列了。Reference (参考)系列作为KEF产品线中一个非常重要的高端系列,历经多次变革,至今依然是KEF产品线当中最受发烧友欢迎的系列。它不仅是嫡传KEF每个时期应用于旗舰级先进技术的产品,更是KEF最新技术的普及应用和向下移植的承载者。这个刚刚迈过第50个年头的经典系列在这么多年来能够持续获得发烧友和媒体的喜爱实属不易,这个系列是如何一步一步成为经典的?我们来回顾一下KEF这个品牌与参考系列的发展。

超级黑胶唱盘New Reference有多硬核? THORENS多能仕总裁Gunter Kurten先生告诉你

德国THORENS(多能仕),自1883年创立以来,已拥有超过140年的辉煌历史,是全球首屈一指的黑胶唱盘品牌之一。值此140周年庆典之际,THORENS于2023年隆重推出了新一代的旗舰产品——New Reference超级黑胶唱盘。

一切皆有可能 测试Consonance Reference CDS8音乐播放器

以前,我从未想过听CD会过时,但在无载体媒体时代,情况已经如此。幸运的是,一群执着的音响发烧友还在坚持。ORERA Consonance Re horee a OPERA Consonance Reference CDS8这款CD、数码流播放器,不仅可以播放CD,而且它是以非常高的水准来播放CD,名副其实的好产品,可以与Advance Acoustics、Creek、AVM等这些品牌的产品齐名。

Design of MC9 Test Equipment for Inertial Reference System

Inertial reference system is one of the airborne equipment.According to the requirements of SAE ARP4754A Guidelines for Development of Civil Aircraft and Systems,MC9 equipment qualification test is needed to verify that the inertial reference system can perform reservation function under specified service conditions.That is,the inertial reference system shall pass certain environmental tests specified in DO⁃160G.Some tests are faced with the problem that the test equipment should have the function requirements of isolation protection and load simulation.Therefore,a kind of test equipment which can provide isolation protection and simulate load function in the test is designed.

The China Seismological Reference Model project

The importance of developing high-resolution seismic models to improve understanding of tectonic processes and enhance seismic hazard mitigation programs,along with the rapid expansion of seismic coverage in China,called for a seismological reference model to be established in China.The China Seismological Reference Model(CSRM)project was initiated by the National Natural Science Foundation of China with two primary goals:(1)the CSRM would serve as a primary source for the current state of seismological research in China,and(2)the seismic data and constraints used to construct the CSRM would be used as a backbone open-access cyberinfrastructure for future research in seismology.The CSRM project was also intended to promote data exchange and scientific collaboration in seismology in China.Accordingly,two parallel efforts of the project are being pursued:(1)construction of the CSRM,and(2)development of a CSRM product center.The CSRM is jointly constrained by various types of seismic constraints extracted from the seismic data recorded at 4511 seismic stations in continental China following a top-down approach,with the seismic structures in the shallower part of the Earth constrained first.Construction of the CSRM involves three preparation steps:(1)building datasets of various seismic constraints from the seismic data,(2)developing a method to incorporate the constraints of surface wave observations from regional earthquakes into the inversion of the seismic structure,and(3)constructing high-resolution pre-CSRM seismic models of the velocity structure in the shallow crust and the Pn-velocity structure in the uppermost mantle.In the final process,the CSRM will be constructed by jointly inverting all the seismic constraints using the pre-CSRM models as starting models or a priori structures.The CSRM product center(http://chinageorefmodel.org)archives and distributes three types of products:CSRM models,the Level 1 original seismic data used to extract seismic constraints in the construction of the CSRM,and Level 2 data on the seismic constraints derived from the Level 1 data and the inferred earthquake parameters in the construction of the CSRM.The CSRM product center has archived 141 TB of Level 1 data from 1120 permanent broadband stations in the China Seismic Network Center and 3391 temporary stations from various institutions and data centers around the world,as well as 140 GB of Level 2 data on various seismic constraints and inferred event parameters from the construction of the CSRM.The CSRM is expected to provide significant insights into the composition and tectonic dynamics in continental China and to enhance the capability of various seismic hazard mitigation programs in China from near real-time rapid determination of earthquake parameters to an earthquake early warning system.The CSRM could also provide guidance for focuses in future seismological research and the design of future active and passive seismic experiments in China.Several focuses are suggested for future seismological research in China,along with the building of a national cyberinfrastructure to sustain and expand the operations of the CSRM project.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

Identification of optimal reference genes in golden Syrian hamster with ethanol-and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction

Background:Acute pancreatitis(AP)is a severe disorder that leads to high morbidity and mortality.Appropriate reference genes are important for gene analysis in AP.This study sought to study the expression stability of several reference genes in the golden Syrian hamster,a model of AP.Methods:AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol(1.35 g/kg)and palmitoleic acid(2 mg/kg).The expression of candidate genes,including Actb,Gapdh,Eef2,Ywhaz,Rps18,Hprt1,Tubb,Rpl13a,Nono,and B2m,in hamster pancreas at different time points(1,3,6,9,and 24 h)posttreatment was analyzed using quantitative polymerase chain reaction.The expression stability of these genes was calculated using Best Keeper,Comprehensive Delta CT,Norm Finder,and ge Norm algorithms and Ref Finder software.Results:Our results show that the expression of these reference genes fluctuated during AP,of which Ywhaz and Gapdh were the most stable genes,whereas Tubb,Eef2,and Actb were the least stable genes.Furthermore,these genes were used to normalize the expression of TNF-αmessenger ribonucleic acid in inflamed pancreas.Conclusions:In conclusion,Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

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 andΔCt 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,EF1αand ACT for floral development;GAPDH,UBCE and ACT for fruit setting;EF1α,GAPDH and eIF2B for fruit ripening;ACT,EF1αand UBCE for leaves under heat stress;eIF2B,UBCE and EF1αfor leaves under freezing stress;EF1α,TUA and UBCE for leaves under salt stress;ACT,EF1αand 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 coldstorage 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.

Maintenance of Millimeter-level Geodetic Reference Framework

The high-precision terrestrial reference frame,as the spatial benchmark for geodesy,is an important national infrastructure.However,due to the influence of nonlinear factors related to geophysical phenomena,the overall maintenance accuracy of the ITRF framework is still at the centimeter level.Therefore,accurately characterizing the true trajectories of linear motion,nonlinear motion,and geocentric motion of the reference station is the key to achieve the construction and maintenance technology of a millimeter level terrestrial reference framework.Based on long-term global and regional GNSS observation data,more Chinese geodesy scientists devoted much efforts to the maintenance of millimeter-level geodetic reference framework.The main contributions of this work included the followings:①Dynamic maintenance of millimeter-level terrestrial reference frame;②Research progress on the method of maintenance of regional reference frame based on GNSS;③The progress of CGCS2000 frame maintenance in millimeter level accuracy;④Reprocessing and reanalysis of two-decade GNSS observation in continental China;⑤Research on current GNSS velocity field model and deformation in Chinese mainland;⑥The preliminary realization and evaluation of CTRF2020.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Responses of vegetation yield to precipitation and reference evapotranspiration in a desert steppe in Inner Mongolia,China

Drought,which restricts the sustainable development of agriculture,ecological health,and social economy,is affected by a variety of factors.It is widely accepted that a single variable cannot fully reflect the characteristics of drought events.Studying precipitation,reference evapotranspiration(ET_(0)),and vegetation yield can derive information to help conserve water resources in grassland ecosystems in arid and semi-arid regions.In this study,the interactions of precipitation,ET_(0),and vegetation yield in Darhan Muminggan Joint Banner(DMJB),a desert steppe in Inner Mongolia Autonomous Region,China were explored using two-dimensional(2D)and three-dimensional(3D)joint distribution models.Three types of Copula functions were applied to quantitatively analyze the joint distribution probability of different combinations of precipitation,ET_(0),and vegetation yield.For the precipitation–ET_(0)dry–wet type,the 2D joint distribution probability with precipitation≤245.69 mm/a or ET_(0)≥959.20 mm/a in DMJB was approximately 0.60,while the joint distribution probability with precipitation≤245.69 mm/a and ET_(0)≥959.20 mm/a was approximately 0.20.Correspondingly,the joint return period that at least one of the two events(precipitation was dry or ET_(0)was wet)occurred was 2 a,and the co-occurrence return period that both events(precipitation was dry and ET_(0)was wet)occurred was 5 a.Under this condition,the interval between dry and wet events would be short,the water supply and demand were unbalanced,and the water demand of vegetation would not be met.In addition,when precipitation remained stable and ET_(0)increased,the 3D joint distribution probability that vegetation yield would decrease due to water shortage in the precipitation–ET_(0)dry–wet years could reach up to 0.60–0.70.In future work,irrigation activities and water allocation criteria need to be implemented to increase vegetation yield and the safety of water resources in the desert steppe of Inner Mongolia.

Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.