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.

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.

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.

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.

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.

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 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.

Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

The accurate measurement of gene expression via quantitative real-time reverse transcription PCR(q RT-PCR)heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA(18S), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), α-tubulin(TUA), β-tubulin(TUB), actin(ACT) and cytochrome oxidase subunit 1(COX1), were evaluated for their expression stability with q RT-PCR and three statistical algorithms(Ge Norm, Norm Finder, and Best Keeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages(resting cysts), different growth stages, in darkness, exposed to abscisic acid(ABA) and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of q RT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.

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.

Identification of reference genes provides functional insights into meiotic recombination suppressors in Gerbera hybrida

Gerbera Hybrida is one of the important cut flowers across the world.The novel traits are the primarily market requirements and the breeding targets,mainly determined by the degree of genetic variation after hybridization.However,meiotic recombination is highly conserved in most eukaryotes which suppressed the crossover formation and limited the genetic diversity.Recently,several meiotic recombination suppressors have been identified and characterized in plants,whereas it remains elusive in G.hybrida.In order to characterize the expression patterns of these suppressors in G.hybrida,20 candidate reference genes were identified from the transcriptome datasets of G.hybrida,and their expression stabilities during plant development were evaluated by geNorm,NormFinder and BestKeeper.Although the most stable reference genes were variable in different softwares,comprehensive ranking revealed that PGK2 was the most stable reference gene and GAPDH was the most unstable one.The expression patterns of FANCM,FIGL1,RECQ4,RM1,and FLIP further validated that PGK2 was suitable for normalization of gene expression.Our study identified a reliable reference gene for gene expression during meiotic recombination,and provided functional insights into meiotic recombination suppressors in G.hybrida.

Transcriptional search to identify and assess reference genes for expression analysis in Solanum lycopersicum under stress and hormone treatment conditions

Tomato(Solanum lycopersicum)is a model plant for research on fruit development and stress response,in which gene expression analysis is frequently conducted.Quantitative PCR(qPCR)is a widely used technique for gene expression analysis,and the selection of reference genes may affect the accuracy of results and even conclusions.Although there have been some frequently used reference genes in tomato,it has been shown that the expressions of some of these genes are not constant in different tissues and environmental conditions.Moreover,little information on genomic identification of reference genes is available in tomato.Here,we mined the publicly available transcriptional sequencing data and screened out fifteen candidate reference genes,and the expression stability of these candidate genes and seven traditionally used ones were evaluated under stress and hormone treatment.The results showed that over half of the selected candidate references were housekeeping genes in tomato cells.Among the candidate reference genes and the traditionally used ones,the most stably expressed genes varied under different treatments,and most of these genes were recommended as preferred reference genes at least once except Solyc04g009030 and Solyc07g066610,two traditionally used reference genes.This study provides some novel reference genes in tomato,and the preferred reference genes under different environmental stimuli,which may be useful for future research.Our study suggests that excavating stably expressed genes from transcriptome sequencing data is a reliable approach to screening reference genes for qPCR analysis.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Paeonia ostii T.Hong et J.X.Zhang Leaves Exposed to Different Drought Stress Conditions

The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy.Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors.In this study,the expression profiles of ten candidate genes,ACT1,ACT2,GAPDH,eIF1,eIF2,α-TUB,β-TUB,TBP,RNA Pol II and RP II,were calculated for a suitable reference gene selection in Paeonia ostii T.Hong et J.X.Zhang leaves under various drought stress conditions.Data were processed by the four regularly used evaluation software.A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one.In addition,the geNorm program provided the optimal choice of two reference gene combination,RNA Pol II andβ-TUB,for qRT-PCR normalization in P.ostii subjected to different drought stress levels.Our research provided convenience for gene expression analysis in P.ostii under drought stress and promoted research of effective methods to alleviate P.ostii drought stress in the future.

Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas oryzae pv.oryzae--infection and melatonin supplementation

Exogenous melatonin(MT)was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv.oryzae(Xoo)-caused bacterial blight(BB).However,the accurate comparison of the expression levels across samples was a challenging task.In this work,the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR(qRT-PCR),and algorithms geNorm,NormFinder and BestKeeper.Our results indicated that most reference genes remained stable in Xoo-infected rice plants,while a number of reference genes were affected by MT supplementation.Among all studied genes,the transcript levels of 18S(18S ribosomal RNA)and UBC(Ubiquitin-conjugating enzyme E2)remained unaltered by Xoo infection,while UBC and UBQ5(Ubiquitin 5)were the most stable genes when examining simultaneous Xoo-infection and MT supplementation,demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice

Accurate quantitative reverse transcription PCR(q RT-PCR)requires reliable reference genes whose expression does not vary in different tissues and developmental stages.However,few reliable reference genes are available for q RT-PCR in rice(Oryza sativa).Here,we established an effective strategy for identifying novel reference genes(NRGs)for reliable normalization of q RT-PCR data in various rice organs and developmental stages.We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database(Rice XPro)data.Genes with low variation(<2.5 cycle quantification)across tissues and developmental stages,and little fluctuation in expression in heatmaps from Rice XPro data were considered stable NRGs.To validate this strategy,we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs,and compared these genes with five established reference genes(ERGs).Only one of the ERGs(UBQ5)was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs.Therefore,public transcriptomic databases are useful for identifying NRGs.We selected two NRGs,UFC1(Homolog of UFM1-Conjugating Enzyme 1)and Fha B(Homolog of Adhesin Fha B)for q RT-PCR analysis in rice;their homologs might be suitable for other monocot plants.

Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in Human Spermatozoa

Objective:Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data.Recently,several strategies have been utilized for validating reference genes in different human tissues.However,no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa.Methods:Using quantitative reverse transcription-polymerase chain reaction(RT-qPCR),we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates.We assessed the stability of reference genes using three different algorithms,namely geNorm,NormFinder,and BestKeeper.We then identified the most stable reference genes.Results:Male-enhanced antigen 1(MEA1)was identified as the most stably expressed reference gene,followed by testis-enhanced gene transcript(TEGT).Conclusions:We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

Screening and validation of reference genes for qPCR analysis in gonads and embryos of Takifugu bimaculatus

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR(qPCR).In this study,the expression of the 10 common candidate reference genes(18s rRNA,rps27,cnbp,rpl7,ube2,hsp-at,gapdh,β-actin,rpl13a,1-ef1a)at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR.And the expression stability of these reference genes is analyzed by GeNorm,NormFinder and Bestkeeper softwares.The results showed that the expression stability of 1-ef1a was the highest(p<0.05)combined with the results of three analysis softwares,and the optimal number of reference genes was 3,which were 1-ef1a,hsp-at and rps27.Based on this result,we analyzed the expression of objective genes(foxl2 and dmrt1)at different developmental stages(early,medium-term and late)of gonads and embryos(eye vesicle stage,motility stage,heart anlage stage,retinal pigmentation stage,newly hatched larva)of T.bimaculatus by qPCR.Among which the highest expression level of foxl2 was in the middle stage of ovary(p<0.05),and dmrt1 was expressed in the early stage of testis with the highest level(p<0.05).The expression levels of foxl2 and dmrt1 in the early stages of embryonic development were low or even almost non-existent,and had a slight increase in the late embryonic development(newly hatched larva).The expression profile of objective genes are consistent with their roles in the development of gonads and embryos,which indicates that 1-ef1a,hsp-at and rps27 are the suitable reference gene combination for detecting the expression of objective genes by qPCR in gonads and embryos of T.bimaculatus.This research also provides the reliability data for screening the reference genes by qPCR in fish.