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.

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.

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 of the appropriate reference genes by quantitative real-time PCR in leopard coral groupers Plectropomus leopardus

Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish species.In this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stability of six common reference genes expression and selected the appropriate reference genes in leopard coral groupers with or without Vibrio harveyi stimulation at different time points.These data produced by qRT-PCR was handled via the geNorm,NormFinder,and BestKeeper software.The results revealed all the examined reference genes had manifest tissue-specific expression in different tissues.Prior to V.harveyi stimulation,RPL13 gene was the appropriate reference gene among eleven tissue types(blood,spleen,hepatopancreas,kidney,stomach,gill,heart,skin,muscle,intestine,brain)in leopard coral groupers.Under V.harveyi stimulation,the most reliable reference genes varied from tissue to tissue and were closely hinged upon different time points.At 6-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were Actin,B2M,UBCE,and Actin,respectively.At 9-and 12-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were RPL13,Actin,Actin,and Actin,respectively.If one reference gene was preferable,RPL13,Actin,Actin,and Actin could be selected as the reference gene in hepatopancreas,spleen,kidney,and gill of leopard coral groupers after V.harveyi infection,respectively.Expression profiles of two target genes(IL-6 and NK-lysin)were used to further validate reliability of these selected appropriate candidates.This study will lay a solid foundation for the future research on qRTPCR analysis of gene expression in leopard coral groupers.

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.

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.

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.

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-1αwas 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.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda(Lepidoptera:Noctuidae)

Real-time quantitative PCR(qPCR)is a reliable and widely used technique for analyzing the expression profiles of target genes in different species,and reference genes with stable expressions have been introduced for the normalization of the data.Therefore,stability evaluation should be considered as the initial step for qPCR experiments.The fall armyworm Spodoptera frugiperda(J.E.Smith)(Lepidoptera:Noctuidae)is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world.However,no studies thus far have examined the stability of reference genes in this pest.In this study,the expression profiles of the eight candidate reference genes of Actin,elongation factor 1 alpha(EF1α),elongation factor 2(EF2),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),ribosomal protein L3(RPL3),ribosomal protein L13(RPL13),alpha-tubulin(α-TUB),and beta-1-tubulin(β-1-TUB)were obtained from S.frugiperda in different samples and the stability was evaluated byΔCt,BestKeeper,geNorm,NormFinder,and RefFinder methods.The results of pairwise variation(V)calculated by GeNorm indicated two reference genes could be selected for normalization.Therefore,the combinations of the most stable reference genes for different experimental conditions of S.frugiperda were shown as follows:EF2 and RPL13 for developmental stages,RPL3 andβ-1-TUB for larval tissue samples,EF2 and EF1αfor the larval samples treated with different temperatures,RPL3 and EF1αfor the larval samples under starvation stress,and RPL13 and EF1αfor all the samples.Our results lay the foundation for the normalization of qPCR analyses in S.frugiperda and could help guarantee the accuracy of subsequent research.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Transcriptome-Wide Identification and Validation of Reference Genes in Black Rockfish(Sebastes schlegelii)

The quantitative real-time reverse transcription PCR(qRT-PCR)is a widely used technique to analyze gene expression levels.Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR.However,most previous studies on fishes adopted reference genes that were commonly used in mammals without validation.In this study,we utilized 89 transcrip-tome datasets covering early developmental stages and different adult tissues,and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii.Finally,121 candidate reference genes were identified based on four criteria.Eight candidates(METAP2,BTF3L4,EIF5A1,TCTP,UBC,PAIRB,RAB10,and DLD)and four commonly used reference genes in mam-mals(TUBA,ACTB,GAPDH,RPL17)were selected for validation via qRT-PCR and four statistical analysis methods(delta-Ct,Best-Keeper,geNorm,and NormFinder).The results indicated that when the black rockfish are cultured in a general condition,the eight candidate reference genes are more stable than traditional reference genes in mammals,and RAB10,EIF5A1,PAIRB and BTF3L4 are the best reference genes in rockfish.This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish,and lay an important foundation for gene expression analysis in teleost.

Identification of suitable reference genes in leaves and roots of rapeseed (Brassica napus L.) under different nutrient deficiencies

Nutrient deficiency stresses often occur simultaneously in soil. Thus, it＇s necessary to investigate the mechanisms underlying plant responses to multiple stresses through identification of some key stress-responsive genes. Quantitative real-time PCR （qRT-PCR） is essential for detecting the expression of the interested genes, of which the selection of suitable reference genes is a crucial step before qRT-PCR. To date, reliable reference genes to normalize qRT-PCR data under different nutrient deficiencies have not been reported in plants. In this study, expression of ten candidate reference genes was detected in leaves and roots of rapeseed （Brassica napus L.） after implementing different nutrient deficiencies for 14 days. These candidate genes, included two traditionally used reference genes and eight genes selected from an RNA- Seq dataset. Two software packages （GeNorm, NormFinder） were employed to evaluate candidate gene stability. Results showed that VHA-E1 was the highest-ranked gene in leaves of nutrient-deficient rapeseed, while VHA-G1 and UBC21 were most stable in nutrient-deficient roots. When rapeseed leaves and roots were combined, UBC21, HTB1, VHA-G1 and A CT7 were most stable among all samples. To evaluate the stabilities of the highest-ranked genes, the relative expression of two target genes, BnTrxl;1 and BnPhtl;3 Were further determined. The results showed that the relative expression of BnTrxl;1 depended on reference gene selection, suggesting that it＇s necessary to evaluate the stability of reference gene prior to qRT-PCR. This study provides suitable reference genes for gene expression analysis of rapeseed responses to different nutrient deficiencies, which is essential for elucidation of mechanisms underlying rapeseed responses to multiple nutrient deficiency stresses

Identification of Suitable Reference Genes for qRT-PCR Normalization in Tilia miqueliana Maxim

Quantitative real-time polymerase chain reaction(qRT-PCR)is a rapid and effective approach toward detecting the expression patterns of target genes.The selection of a stable reference gene under specific test condition is essential for expressing levels of target genes accurately.Tilia miqueliana,considered endangered,is a prominent native ornamental and honey tree in East China.No study has evaluated the optimal endogenous reference gene for qRT-PCR analysis in T.miqueliana systematically.In this study,fifteen commonly used reference genes were selected as candidate genes,and the stabilities of their expressions were assessed using four algorithms(GeNorm,NormFiner,BestKeeper,and DeltaCt)in nine experimental datasets.The final integrated evaluation was performed using a comprehensive analysis algorithm(RefFinder).Finally,a target MYB transcription factor gene(TmMYB)was used to verify the accuracy of the candidate reference genes.The results showed that PP2αwas the most stable in tissue set,while RPS13 and SAMCD were optimal for heat and cold stress,respectively.Under waterlogging stress,PP2αand TUB were the most stable genes in the leaves and roots,respectively.EF1αand PP2αwere optimal for drying stress in leaf and root tissues.TUB and ACT7 were the most stable genes in the leaf and root tissues under salt stress.This is the first systematic evaluation of candidate reference genes in T.miqueliana,and it will benefit future studies on expression and functional analysis of target genes in T.miqueliana.

Selection and Verification of Reference Genes for qRT-PCR Analysis in Iris domestica under Drought

Iris domestica is a plant of the Iridaceae family and is drought-tolerant,but its drought-resistance mechanism is not yet clear.Analysing the gene expression changes of I.domestica by qRT-PCR is an important mean to understand its drought resistance characteristics.Nevertheless,a lack of reference genes greatly hinders investigation and research on the adaptation of I.domestica to drought at the molecular and genetic levels.In this study,we assessed the expression stability of 11 candidate gene in I.domestica under drought stress conditions and different tissues using geNorm,NormFinder,BestKeeper and RefFinder tools.The results showed that EF1βwas the most stable reference genes under drought stress and in different tissues.To validate further the stability of the identified reference genes,the expression patterns of VP gene in I.domestica was analysed.These results will be conducive to more accurate quantification of gene expression levels in I.domestica.

Selection of Reference Genes in Saccharopolyspora Spinosa for Real-Time PCR

Reverse transcription quantitative PCR （RT-qPCR） combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene（sl. The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Screening of stable internal reference genes by quantitative real-time PCR in humpback grouper Cromileptes altivelis

Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV)through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

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.