Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.

Detection of gemcitabine-resistant genes expression in lung cancer cell lines using real-time PCR

Objective: To explore the molecular mechanism of gemcitabine-resistance, the relative mRNA expression of five genes related to gemcitabine-resistance was detected in six lung cancer cell lines. Methods: The total RNA was extracted from six lung cancer cell lines GLC-82, NCI-H460, A549, 95-C, 95-D and QG56. Then the cDNA was amplified by real-time quantitative PCR method to quantify the gene expression of RRM1, PTEN, ERCC1, dCK and CDA. The cytotoxicity of gem- citabine to cell lines was tested by MTT method. Results: Among the detected six lung cancer cell lines, the mRNA level of RRM1, PTEN and ERCC1 in lung squamous cell line QG56 was highest, and the IC50 of gemcitabine to QG56 cell line was also highest. Conclusion: The mRNA expression of RRM1, PTEN and ERCC1 was correlated, and the high expression of RRM1 was related to gemcitabine resistance of lung cancer.

Insecticidal Potential of α-Pinene and β-Caryophyllene against Myzus persicae and Their Impacts on Gene Expression

Myzus persicae(M.persicae)is now considered a threat to agricultural crops due to economic losses.Numerous synthetic insecticides applied every year against M.persicae,are reported to be unsafe for environment,humans,and beneficial insects.Furthermore,several species of Myzus have been found to develop resistance due to over application of these insecticides.Therefore,it is required to find some novel insecticide that would be safe for the environment as well as for humans.In the current study,two major pure constituentsα-pinene andβ-caryophyllene were evaluated for their insecticidal potential against M.persicae using a fumigant toxicity assay.Furthermore,impact ofα-pinene andβ-caryophyllene on expression of five different genes,e.g.,HSP 60,FPPS I,OSD,TOL and ANT responsible for reproduction,dispersion,and growth of M.persicae has also been investigated.To perform fumigant toxicity assay,five different concentrations(3.5,4,4.5,5 and 6μL L−1)ofα-pinene andβ-caryophyllene were prepared.Lethal concentration(LC)was calculated,and gene expression studies were executed through qRT PCR at LC30 ofα-pinene andβ-caryophyllene.Both constituents demonstrated excellent fumigant toxicity effects against M.persicae at all five concentrations.However,α-pinene shows significantly better results(98%)as compared toβ-caryophyllene(80%)after 72 h at 6μL L−1 of dose.The highest upregulation in expression was demonstrated at LC30 dose ofα-pinene in five in three out of five genes understudy(TOL,ANT,and FPPS I).Conversely,two genes HSP 60 and OSD demonstrated downregulation at LC30 dose ofβ-caryophyllene.Conclusively,our results highlighted the promising insecticidal potential of both compoundsα-pinene andβ-caryophylleneby interfering with the reproduction and development related processes in M.persicae,allowing us to recommend the phytoconstituents under investigation as an ecofriendly alternative to synthetic insecticides.

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.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

DDRT-PCR Analysis of Wheat Roots Under Iron-Deficient Condition and Differential Expression of ABC Gene

Differential expression of gene in iron-efficient wheat cultivar Jing411 and iron-inefficient cul-tivar SanshumaiS under iron-deficiency and iron-sufficiency conditions was revealed by differential display reverse transcript PCR (DDRT-PCR) method. Northern blotting was carried out using ATP-binding transporter (ABC) cDNA obtained from DDRT-PCR products of the cultivar Jing411 as probe. Our results suggested that ABC gene expression was suppressed under iron-deficiency condition.

Analysis of Gene Expression of Seven Isoforms of ADP-glucose Pyrophosphorylase in Rice Endosperm under Different Temperature Conditions

[Objective] This study aimed to analyze the effects of temperature on the expression of AGPase isoform genes in rice endosperm during milk stage. [Method] Different temperature treatments （33 and 25 ℃ of daily mean temperature for high and normal temperature treatments, respectively） and the real-time fluorescence quantitative PCR （ FQPCR） were used to analyze the expression patterns of seven isoforms （AGPS1, AGPS2a, AGPS2b, AGPL1, AGPL2, AGPL3 and AGPL4） of ADPglucose pyrophosphorylase （AGPase） which was the key enzyme in starch synthesis and metabolism in rice endosperm of two rice varieties Teqing and Thai Fragrant Rice. [Result] The AGPase isoforms AGPS2b, AGPL2 and AGPL3 had much higher expression than the other four isoforms, thus they were thought to be the main expression patterns of AGPase in rice endosperm. The relative expressions of AGPL2 was the highest among all the isoforms. The relative expressions of AGPS2b, AGPL2 and AGPL3 were higher in the normal temperature treatment than in the high temperature treatment in both rice varieties. The relative expression of the three enzyme genes in milk stages in Teqing was higher than those in Thai Fragrant Rice under different temperature treatments. [Conclusion] This study provides a theoretical basis for further use of molecular biology techniques to cultivate stable high-quality rice varieties.

Use of Real- time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 cleady and similady, namely NtFer1 expression was responsive to iron-ovedoad, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

Enhancement of NH_4^+ Uptake by NO_3^- in Relation to Expression of Nitrate-Induced Genes in Rice (Oryza sativa) Roots

This study aimed to survey the expression of genes involved in rice N uptake and aasimilatory network and to understand the potential molecular mechanisms responsible for the NO3^-enhanced NH4^＋ uptake. By using quantitative real-time polymerase chain reaction （PCR）, the genes related to N nutrition, including ammonium transporters （AMTs） and ammonium assimilatory enzymes （GS and GOGAT）, were transcriptionally analyzed in rice plants grown in the absence and presence of NO4^- in the NH4^＋-containing medium. The results showed that NH4^＋ uptake by rice was enhanced by the NO3^- supply to the medium. At the same time and in parallel, the amount of transcripts of seven genes （OsAMT1;1, OsAMT1;2, OsAMT4;1, OsGLNP, OsGLU1, OsGLT1, and OsGLTP） was increased in rice roots, but the expression of two genes （OsGLN1;1 and OsGLN1;P） was decreased and that of OsAMT1;3 remained without change. Up- or downregulation of these genes involved in NH4^＋ uptake and assimilation correlated with the increase in NH4^＋ uptake in the presence of NO3^- in rice roots.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Effects of trichostatin A(TSA)on growth and gene expression in HeLa cells

Objective： To investigate the expressions of p53, RB1, Fas, c-fos, Ras, EGFR mRNA in human cervical cancer （HeLa） cell in response to the trichostatin A （TSA）. Methods： We took count of HeLa cells in different incubation times with TSA （0.2μm/L）. The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h. Then, we investigated the genes expression （mRNA levels） of HeLa cells after treatment for 36 h using SYBR green real-time PCR. Results： We demonstrated that trichostatin A （TSA） could make human cervical cancer （HeLa） cell morphological change and induce HeLa cell apoptosis. Furthermore, the data suggest that TSA-induced down-regulation of p53, RB1, Fas, but upregulated c-fos gene expression after treatment for 36 h, and Ras, EGFR did not show obvious response to TSA treatments. Conclusion： TSA has different effects on gene expression.

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display (DDRT-PCR) technique was adopted to find out the genes related tosettlement metamorphosis development process of Ruditapes philippinarum larvae.In this study,we haveobtained three hundred and forty-six amplification bands in total from pediveliger larvae,veliger larvae,eye spot larvae and post-larvae.Sixty-five out of three hundred and forty-six bands are distinctly differen-tial display from band pattern,which can be put into four groups,standing for different expression char-acters.Sixteen differential display bands were cloned,sequenced and analyzed and nine different se-quences are obtained in the study.Three sequences have higher similarity to the cDNAs deposited indatabase and three are very similar to the rDNA of other species,considered as the rDNA of Ruditapesphilippinarum.The rest three sequences are found to be novel sequences after analyzed.Their accessionnumbers are AY916799,AY916798,and AY916797 respectively.We thought the novel sequences arepossibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide somefundamental understandings that are helpful for the improvement of scallop seed raising industry.

Quantification of the expression of chitinolytic enzyme encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions using a real-time RT-PCR assay

The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.

Effects of Wei Chang An on expression of multiple genes in human gastric cancer grafted onto nude mice

AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafted onto nude mice was used as the animal model. The mice were randomly divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil （5-FU） over 6-d period starting at 8th d after grafting. Control animals received saline on an identical schedule. Animals were killed 41 d after being grafted. The expression profiles in paired WCA treated gastric cancer samples and the N.S. control samples were studied by using a cDNA array representing 14181 cDNA clusters. The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction （qPCR）. RESULTS： When compared with controls, the average tumor inhibitory rate in WCA group was 44.32% ± 5.67% and 5-FU 47.04% ± 22.33% （P 〈 0.01, respectively）. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group. Apoptotic index （AI） was significantly increased to 9.72% ± 4.52% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method in WCA group compared with the controls 2.45% ± 2.37%. 5-FU group was also found to have a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expressed sequence tags （ESTs） among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. By using qPCR, the expression level of Stat3, rap2 interacting protein x （RIPX）, regulator of differentiation 1 （ROD1） and Bcl-2 was lower in WCA group than that in control group respectively. By using SP immunohistochemical method the expression of Phospho-Stat3 （Tyr705） and Bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively. CONCLUSION： WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Star3, RIPX, ROD1 and Bcl-2 gene.

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 （RACK1） is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference （RNAi）, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

CLONING AND EXPRESSION OF Fab GENES OF ANTI-STOMACH CANCER McAb 3G9 IN E. COLI

Immunoglobulin heavy chain Fd gene and K chain gene were amplified by Polymerase Chain Reaction (PCR) from total RNA of a hybridom acell line secreting anti-stomach cancer monoclonal antibody 3G9. Sequences analysis showed that VH, D, JH genes were derived from VH7183, DFL16. 1, and JH3, and that V. and J, from VkⅡ and Jk2.3G9 Fd and K DNA segments were cloned Into expression vector pComb3. Soluble Fab was expressed by IPTG Induction and proved by western blotting. The specific antigen binding activity of the bacterially produced 3G9 Fab was demonstrated by enzyme- linked Immunofiltration

Isolation and Expression Analysis of MaPRMT1 Gene in Banana

[Objective] The aim of experiment was to lay molecular foundation for studying maturity mechanism of banana after harvest. [Method] The combined method of suppressing subtractive hybridization and cDNA micro-array were used to obtain cDNA segment of one PRMT gene in banana and the whole cDNA sequence of the gene was cloned.The bioinformatics analysis was operated on it,in addition, the expression profile analysis was conducted in different organs and different mature periods of banana.[Result] The whole length of cDNA in MaPRMT1 was 1 158 bp and possessed a complete open reading frame,which could encode 385 amino acids.It had high homology with PRMT in plant,containing one Methyltransf_1 domain.The MaPRMT1 gene was expressed in root,stem,leaf and fruit of banana and the expression levels in stem and leaf were relatively high.As the increase of days after harvest,the expression level declined gradually,however it reached maximum when ethylene release was biggest,then it declined.[Conclusion] MaPRMT1 belonged to the first kind of arginine methyltransferase and it was expressed differently in different organs and fruits at different mature periods.

Association between IL-37 and VEGF Gene Expression in Psoriasis Pathogenesis in Egyptian Population

Background: Psoriasis is considered a common skin disease, marked by the production and elevation of inflammatory plaques that regularly shed scales resulting from extensive skin epithelial cell proliferation. T lymphocytes, neutrophils, and other leukocytes enter the irritated skin, resulting in epidermal keratinocyte hyperplasia, vascular hyperplasia, ectasia, and T lymphocyte, neutrophil other forms of leukocyte infiltration. Aim of the Work: the study aimed to investigate the role of IL-37 and VEGF gene expression in the pathogenesis of psoriasis in Egyptian patients. Methodology: Polymerase chain reaction techniques (PCR) were applied to detect VEGF in the skin homogenates of psoriasis patients. In addition, the ELIZA technique was applied to investigate IL-37 in skin homogenates. One hundred cases have been divided into two groups: 50 healthy volunteers as the group I healthy control;50 psoriasis patients as group II. PCR real-time technology was assessed through extracted DNA samples for VEGF amplification in skin homogenate. Result: Our results revealed that psoriasis patients significantly had a substantial reduction in IL-37 compared to the control group (p Conclusion: There is a significant inverse association between IL-37 and VEGF in psoriasis patients. Study findings revealed that IL-37 gene expression decreases while VEGF gene expression increases in psoriatic individuals. Such a measurement may be beneficial in determining the severity of the condition, as well as taking into consideration of the disease’s diagnosis.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.