Real-time PCR Data Research Articles

Abstract 665: Perivascular Adipose Tissue near Aortic Arch is a Major Site for Atheroprotective IgM Producing B-1 Cells

Background: Perivascular adipose tissue (PVAT) regulates artery physiology and pathology, such as promoting atherosclerosis development through local production of inflammatory cytokines. The phenotype of PVAT is region-specific and PVAT composed of brown adipose tissue (near the aortic arch and thoracic aorta) and white adipose tissue (near the abdominal aorta). B-1 cells limit adipose tissue inflammation and atherosclerosis through the production of IgM antibodies. PVAT harbors high numbers of B cells. Id3 is a basic helix-loop-helix protein and important for B cell development. B cell-specific Id3 deficiency (Id3 BKO ) increases B-1b cell numbers and provides atheroprotection. However, the location and regulation of IgM producing B-1 cells in the PVAT are unknown. Methods and Results: Flow cytometry analysis of PVAT of normal chow diet fed young ApoE -/- mice (n=5) demonstrated that the abundant CD19 + B cells (per gram fat) harbored in PVAT around aortic arch (2.7 + 0.69 x10 5 cells) compared to PVAT near the thoracic (0.9 + 0.29 x10 5 cells) and abdominal aorta (1.1 + 0.51 x10 5 cells). Interestingly, a large proportion (40%) of these B cells are belong to the CD19 hi B220 low B-1 subset in PVAT near aortic arch. CXCL13 is a chemokine, which is important for B-1 cell recruitment to omental fat. Real time-PCR data confirmed that high numbers of B-1 cell recruitment to PVAT near aortic arch is due to high expression of CXCL13 in the PVAT near the aortic arch compared to PVAT near thoracic aorta and abdominal aorta. Moreover, Flow cytometry and ELISPOT data in normal chow fed young ApoE -/- mice with Id3 BKO demonstrated that Id3 BKO increased B-1b cells (Id3 BKO : 1.2 + 0.16 x10 3 ; Id3 WT : 0.4 + 0.06 x10 3 ; p-value: <0.01; n=6 mice/group) and IgM secreting cells (Id3 BKO : 2.0 + 0.42 x10 3 ; Id3 WT : 0.78 + 0.23 x10 3 ; p-value: <0.05; n=6 mice/group) respectively in PVAT near aortic arch compared to Id3 WT control mice. Conclusion: Results provide the first evidence that atheroprotective B-1 cell profile in PVAT is region-specific and identify Id3 and CXCL13 as regulators of aortic arch PVAT B-1b cell numbers and IgM production.

Cardiogenic effects of characterized Geum urbanum extracts on adipose-derived human mesenchymal stem cells.

Stem cell therapy is considered as a promising treatment for cardiovascular diseases. Adipose-derived mesenchymal stem cells (ADMSCs) have the ability to undergo cardiomyogenesis. Medicinal plants are effective and safe candidates for cell differentiation. Therefore, the aim of our study was to investigate cardiogenic effects of characterized (HPLC-UV) extracts of Geum urbanum on ADMSCs of adipose tissue. The methanolic extracts of the root and aerial parts of G. urbanum were obtained and MTT assay was used for studying their cytotoxic effects. Then, cells were treated with 50 or 100 μg/mL of the extracts from root and aerial parts of G. urbanum. MTT assay showed that the extracts of G. urbanum did not have any toxic effects on ADMSCs. Immunostaining results showed increase in the expression of α-actinin and cardiac troponin I (cTnI), and quantitative real-time reverse-transcription PCR data confirmed the upregulation of ACTN, ACTC1, and TNNI3 genes in ADMSCs after treatment. According to HPLC fingerprinting, some cardiogenic effects of G. urbanum extracts are probably due to ellagic and gallic acid derivatives. Our findings indicated that G. urbanum extracts effectively upregulated some essential cardiogenic markers, which confirmed the therapeutic role of this plant as a traditional cardiac medicine.

GA3 application in grapes (Vitis vinifera L.) modulates different sets of genes at cluster emergence, full bloom, and berry stage as revealed by RNA sequence-based transcriptome analysis

In grapes (Vitis vinifera L.), exogenous gibberellic acid (GA3) is applied at different stages of bunch development to achieve desirable bunch shape and berry size in seedless grapes used for table purpose. RNA sequence-based transcriptome analysis was used to understand the mechanism of GA3 action at cluster emergence, full bloom, and berry stage in table grape variety Thompson Seedless. At cluster emergence, rachis samples were collected at 6 and 24h after application of GA3, whereas flower clusters and berry samples were collected at 6, 24, and 48h after application at full bloom and 3-4mm berry stages. Seven hundred thirty-three genes were differentially expressed in GA3-treated samples. At rachis and flower cluster stage respectively, 126 and 264 genes were found to be significantly differentially expressed within 6h of GA3 application. The number of DEG reduced considerably at 24h. However, at berry stage, major changes occurred even at 24h and a number of DEGs at 6 and 24h were 174 and 191, respectively. As compared to upregulated genes, larger numbers of genes were downregulated. Stage-specific response to the GA3 application was observed as evident from the unique set of DEGs at each stage and only a few common genes among three stages. Among the DEGs, 67 were transcription factors. Functional categorization and enrichment analysis revealed that several transcripts involved in sucrose and hexose metabolism, hormone and secondary metabolism, and abiotic and biotic stimuli were enriched in response to application of GA3. A high correlation was recorded for real-time PCR and transcriptome data for selected DEGs, thus indicating the robustness of transcriptome data obtained in this study for understanding the GA3 response at different stages of berry development in grape. Chromosomal localization of DEGs and identification of polymorphic microsatellite markers in selected genes have potential for their use in breeding for varieties with improved bunch architecture.

Reference gene selection for molecular studies of dormancy in wild oat (Avena fatua L.) caryopses by RT-qPCR method.

Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α), AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.

Conjunction of G-quadruplex and stem-loop in the 5' untranslated region of mouse hepatocyte nuclear factor 4-alpha1 mediates strong inhibition of protein expression.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a well-established master regulator of liver development and function. Restoration of HNF4α can treat multiple liver disorders and liver cancers. To date, HNF4α is still "undruggable" due to lack of known activating ligands. Thus, understanding the regulatory mechanism of HNF4α expression may help develop an alternative approach to modulate HNF4α protein levels. G-quadruplexes (G4) are non-canonical stable secondary structures discovered mostly in the promoters of oncogenes. Recent genome-wide studies demonstrate the enrichment of G4s in the 5' untranslated region (UTR). By protoporphyrin IX-binding assay and circular dichroism spectrum, we validated the presence of a chemically highly stable 4-ring G4 within the 5' UTR of mouse Hnf4a1. Our real-time PCR and Western blot data showed that the Hnf4a1 5' UTR caused a remarkable translational suppression regardless of a moderate effect on Hnf4a1 mRNA levels. The subsequent deletion/mutation analysis of Hnf4a1 5' UTR using dual-luciferase reporter assays further demonstrated that although the disruption of the chemically highly stable 4-ring G4 resulted in a marked attenuation of inhibition, the G4 alone only weakly inhibited translation. Likewise, disruption of a long stem-loop adjacent to the 4-ring G4 markedly attenuated translational inhibition, although the stem-loop alone only exerted a weak inhibitory effect. Thus, the tight conjunction of G4s and an adjacent stem-loop within the Hnf4a1 5' UTR was both necessary and sufficient to mediate the very strong translational repression. Our results establish a novel working model that a chemically stable G4 may require co-factors to be bio-stable for exerting biological functions.

A Rare Rs139365823 Polymorphism in Pre-miR-138 Is Associated with Risk of Congenital Heart Disease in a Chinese Population.

miR-138 modulates cardiac morphogenesis in zebrafish. We explored whether a genetic polymorphism in miR-138 might contribute to the occurrence of sporadic congenital heart disease (CHD) and the potential mechanism. We performed a case-control study consisting of 857 CHD cases and 938 non-CHD controls by genotyping miR-138 in a Chinese population. Two SNPs, including rare rs139365823 located in the pre-miR-138 sequence and rs76987351 located in the pri-miR-138 sequence, were identified by sequencing miR-138. The results demonstrated that the genotypes and allele frequencies of the rs139365823 minor allele A were significantly associated with the increased risk of CHD cases overall or in the Tetralogy of Fallot (TOF) subtype, but not with the rs76987351 A/G allele. Real-time PCR data showed that the rs139365823 minor allele A significantly increased the expression of mature miR-138, whereas the rs76987351 minor allele A had the opposite effect. As TOF is caused by severe outflow tract (OFT) development and an alignment defect, we identified Dvl2, involved in OFT development, as a direct target of miR-138. Further, the rs139365823 minor allele A enhanced the miR-138-mediated inhibitory regulation of Dvl2. Taken together, our results demonstrated for the first time that the functional variant rs139365823 in pre-miR-138 altered the expression of mature miR-138 and its inhibitory effect on target genes and conferred the risk for CHD in the population studied here.

Prognostic Implication of Urothelial Stem Cell Markers Differs According to Primary Tumour Location in Non-Muscle-Invasive Bladder Cancer

Background/Aims: This study aimed to validate the value of urothelial stem cell (USC) markers ΔNp63, integrin β4, CD47, and CD44v6 in predicting the prognosis of non-muscle invasive bladder cancer (NMIBC) located in different anatomic regions of bladder. Methods: The study reviewed the clinicopathologic data of 169 patients with NMIBC. Using real-time PCR and immunohistochemistry, the expression of ΔNp63, integrin β4, CD47, and CD44v6 in archived specimens of patients with NMIBC were validated. Kaplan–Meier analysis and Cox proportional hazards model were used to assess the prognostic impact of USC markers for recurrent-free survival (RFS). Results: The Real-time PCR data showed that the expression of USC markers were higher in tumors located in the trigone and posterior wall than that in other regions of bladder (P< 0.05). Statistical analysis showed that high expression of ΔNp63 was correlated with tumor stage (P=0.023) and tumor size (P=0.001), that high expression of integrin β4 was correlated with tumor stage (P=0.026), tumor grade (P=0.005) and tumor size (P=0.003), and that high integrin β4, CD47, and CD44v6 expression were significantly associated with tumor recurrence (P=0.032, P=0.010, and P=0.043, respectively). Moreover, high expression of ΔNp63 and integrin β4 was correlated with poor RFS in patients with tumors located in the trigone (P=0.025 and P=0.023, respectively). High expression of integrin β4, CD47, and CD44v6 was correlated with poor RFS in patients with tumors in the posterior wall (P=0.017, P=0.033 and P=0.047, respectively). High expression of integrin β4 and CD47 was correlated with poor RFS in patients with tumors in the trigone/posterior wall area (P=0.002 and P=0.005, respectively). Conclusion: Our results suggest that USC markers are linked with poor prognosis of NMIBC patients, especially in patients with tumors in the trigone and posterior wall.

Free Circulating miRNAs Measurement in Clinical Settings: The Still Unsolved Issue of the Normalization.

Circulating molecules that are released into the circulation in response to specific stimuli are considered potential biomarkers for physiological or pathological processes. Their effective usefulness as biomarkers resides in their stability and high availability in all the biological fluids, combined with the limited invasiveness of intervention. Among the circulating molecules, miRNAs represent a novel class of biomarkers as they possess all the required characteristics such as sensitivity, predictivity, specificity, robustness, translatability, and noninvasiveness.miRNAs are small non-coding RNAs, that act as inhibitors of protein translation, and intervene in the complex network of the post-transcriptional mechanisms finely regulating gene expression.The emerging role of miRNAs as potential biomarkers for clinical applications (e.g., cancer and cardiovascular diseases diagnosis and prediction, musculoskeletal disease diagnosis and bone fracture risk prediction), however, requires the standardization of miRNA processing, from sample collection and sample storage, to RNA isolation, RNA reverse-transcription, and data analyses. Normalization is one of the most controversial issues related to quantitative Real-Time PCR data analysis since no universally accepted normalization strategies and reference genes exist, even more importantly, for circulating miRNA quantification. As it is widely demonstrated that the choice of different normalization strategies influences the results of gene expression analysis, it is important to select the most appropriate normalizers for each experimental set. This review discloses on the different strategies adopted in RT-qPCR miRNA normalization and the concerning issues to highlight on the need of a universally accepted methodology to make comparable the results produced by different studies.

Host gut-derived probiotic bacteria promote hypertrophic muscle progression and upregulate growth-related gene expression of slow-growing Malaysian Mahseer Tor tambroides

In modern aquaculture, dietary supplementation of probiotics is a novel approach for enhancing growth performance of slow-growing fish. However, the actual role of probiotics in regulating muscle growth at cellular and molecular levels in fish still needs to be clarified. In this study, we hypothesized that host gut derived probiotic bacteria would enhance cellular muscle growth, and upregulate growth-related gene expression in slow-growing Malaysian mahseer Tor tambroides. Therefore, three host-associated probiotics (Bacillus sp. AHG22, Alcaligenes sp. AFG22, and Shewanella sp. AFG21) were isolated from the gastro-intestinal tract of T. tambroides and screened based on their digestive enzyme activity. A fishmeal and casein based control diet (40% crude protein and 10% lipid) was formulated, and three different probiotic supplemented diets were prepared by immersing the control diet in each isolated host-derived bacteria, suspended in sterile phosphate buffered saline (PBS), to achieve a final concentration of approximately 1.0 × 108 CFU g−1 feed. Triplicate groups of T. tambroides juveniles (initial weight 1.39 ± 0.06 g) were stocked in twelve glass aquaria (100 L capacity) with stocking density of 20 individuals per aquarium. The feed was applied twice daily at 3.0% of the fish body weight per day for 90 days. Growth performance (weight gain and specific growth rate) of T. tambroides juveniles were significantly higher in Alcaligenes sp. AFG22 and Bacillus sp. AHG22 supplemented diet treatments. Muscle morphometric analysis revealed that dietary supplementation of host-associated probiotic bacteria did not influence the frequency distribution of hyperplastic (class 10) small diameter fibers (≤10 μm). However, hypertrophic (Class 50, Class 60 and Class 70) large diameter fibers (>50 μm) were significantly higher in Alcaligenes sp. AFG22 and Bacillus sp. AHG22 supplemented treatments, indicating that increased growth rate of T. tambroides in these treatments was mostly governed by increased muscle fibers hypertrophy, rather than by hyperplasia. Real-time PCR data demonstrated that the relative mRNA expression of GH and IGF1 was upregulated in juvenile T. tambroides fed the diets supplemented with Alcaligenes sp. AFG22 and Bacillus sp. AHG22. These results indicate that host-derived probiotics, especially Alcaligenes sp. AFG22 and Bacillus sp. AHG22, have a significant potential as autochthonous probiotics for the stimulation of growth performance of slow-growing T. tambroides in aquaculture.

Deletion of Cdc42 in embryonic cardiomyocytes results in right ventricle hypoplasia

BackgroundCdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cytoskeleton remodeling and cell polarity establishment. Inactivating Cdc42 in cardiomyocytes resulted in embryonic lethality with heart developmental defects, including ventricular septum defects and thin ventricle wall syndrome.FindingsIn this study, we have generated a Cdc42 cardiomyocyte knockout mouse line by crossing Cdc42/flox mice with myosin light chain 2a (MLC2a)-Cre mice. We found that the deletion of Cdc42 in embryonic cardiomyocytes resulted in an underdeveloped right ventricle. Microarray analysis and real-time PCR data analysis displayed that the deletion of Cdc42 decreased dHand expression level. In addition, we found evaginations in the ventricle walls of Cdc42 knockout hearts.ConclusionWe concluded that Cdc42 plays an essential role in right ventricle growth.

Development of an in vitro model to study uterine functions and early implantation using rat uterine explants.

This study was conducted to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2mm) were isolated, cultured and further characterized. Steroid hormone treatment of cultured explants showed that both Muc1 and Pr were significantly up-regulated (P<0.05) by E2. Areg was significantly up-regulated (P<0.05) by P4 and Igfbp1 was significantly up-regulated (P<0.05) by the combination of E2 and P4, although, in rat, Igfbp1 is E2-dependent. In vitro decidualization of cultured explants was induced and two potential markers of decidualization, Prl8a2 and Bmp2, were examined. Real-time quantitative PCR data revealed that both Prl8a2 and Bmp2 were significantly up-regulated (P<0.05) in MPA- and db-cAMP-treated explants compared to the control group of explants. Then, an individual hatched blastocyst and cultured explant was placed in a 96-well(round-bottom U-shaped) plate. Co-culture results showed that stable attachments were observed after 48h, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. The rates of attachment of embryos to the explants were increased significantly in the P4-treated group (63.6%) compared to the control group (35.5%), after steroid hormone treatment. The rates of attachment were reduced significantly in the E2-treated group (0.0%), where no stable attachments were observed. Despite the necessity of comprehensive investigation, our results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation.

MKK4 from Litopenaeus vannamei is a regulator of p38 MAPK kinase and involved in anti-bacterial response

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment.

Cingulate Alpha-2A Adrenoceptors Mediate the Effects of Clonidine on Spontaneous Pain Induced by Peripheral Nerve Injury.

The anterior cingulate cortex (ACC) is an important brain area for the regulation of neuropathic pain. The α2A adrenoceptor is a good target for pain management. However, the role of cingulate α2A adrenoceptors in the regulation of neuropathic pain has been less studied. In this study, we investigated the involvement of cingulate α2A adrenoceptors in the regulation of neuropathic pain at different time points after peripheral nerve injury in mice. The application of clonidine, either systemically (0.5 mg/kg intraperitoneally) or specifically to the ACC, increased paw withdrawal thresholds (PWTs) and induced conditioned place preference (CPP) at day 7 after nerve injury, suggesting that cingulate α2 adrenoceptors are involved in the regulation of pain-like behaviors. Quantitative real-time PCR data showed that α2A adrenoceptors are the dominant α2 adrenoceptors in the ACC. Furthermore, the expression of cingulate α2A adrenoceptors was increased at day 3 and day 7 after nerve injury, but decreased at day 14, while no change was detected in the concentration of adrenaline or noradrenaline. BRL-44408 maleate, a selective antagonist of α2A adrenoceptors, was microinfused into the ACC. This blocking of cingulate α2A adrenoceptors activity abolished the CPP induced by clonidine (0.5 mg/kg intraperitoneally) but not the effects on PWTs at day 7. However, clonidine applied systemically or specifically to the ACC at day 14 increased the PWTs but failed to induce CPP; this negative effect was reversed by the overexpression of cingulate α2A adrenoceptors. These results suggest that cingulate α2A adrenoceptors are necessary for the analgesic effects of clonidine on spontaneous pain.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of dysfunctional pollen. Based on reliable reference gene-normalized real-time quantitative PCR (RT-qPCR) data, examining gene expression profile can provide valuable information on the molecular mechanism of kenaf CMS. However, studies have not been conducted regarding selection of reference genes for normalizing RT-qPCR data in the CMS and maintainer lines of kenaf crop. Therefore, we studied 10 candidate reference genes (ACT3, ELF1A, G6PD, PEPKR1, TUB, TUA, CYP, GAPDH, H3, and 18S) to assess their expression stability at three stages of pollen development in CMS line 722A and maintainer line 722B of kenaf. Five computational statistical approaches (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) were used to evaluate the expression stability levels of these genes. According to RefFinder and GeNorm, the combination of TUB, CYP, and PEPKR1 was identified as an internal control for the accurate normalization across all sample set, which was further confirmed by validating the expression of HcPDIL5-2a. Furthermore, the combination of TUB, CYP, and PEPKR1 was used to differentiate the expression pattern of five mitochondria F1F0-ATPase subunit genes (atp1, atp4, atp6, atp8, and atp9) by RT-qPCR during pollen development in CMS line 722A and maintainer line 722B. We found that atp1, atp6, and atp9 exhibited significantly different expression patterns during pollen development in line 722A compared with line 722B. This is the first systematic study of reference genes selection for CMS and will provide useful information for future research on the gene expressions and molecular mechanisms underlying CMS in kenaf.

Enabling reproducible real-time quantitative PCR research: the RDML package.

Reproducibility, a cornerstone of research, requires defined data formats, which include the setup and output of experiments. The real-time PCR data markup language (RDML) is a recommended standard of the minimum information for publication of quantitative real-time PCR experiments guidelines. Despite the popularity of the RDML format for analysis of quantitative PCR data, handling of RDML files is not yet widely supported in all PCR curve analysis softwares. This study describes the open-source RDML package for the statistical computing language R. RDML is compatible with RDML versions ≤ 1.2 and provides functionality to (i) import RDML data; (ii) extract sample information (e.g. targets and concentration); (iii) transform data to various formats of the R environment; (iv) generate human-readable run summaries; and (v) to create RDML files from user data. In addition, RDML offers a graphical user interface to read, edit and create RDML files. https://cran.r-project.org/package=RDML. rdmlEdit server http://shtest.evrogen.net/rdmlEdit/. Documentation: http://kablag.github.io/RDML/. k.blag@yandex.ru. Supplementary data are available at Bioinformatics online.

Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula

ObjectiveTo identify reliable reference genes (RGs) for normalization of real-time PCR (RT-qPCR) data in Codonopsis pilosula. MethodsThe expression profiles of 10 candidate RGs (GAPDH, ACT, α-TUB, β-TUB, UBQ, CYP, EF-1α, NAC, F-box and PP2A) were examined in C. pilosula during the phenological period. The raw materials examined included roots, stems, leaves, and flower buds at flowering and boll-forming stages, five growth stages of untreated and treated roots with plant growth retardant. The best-suited RGs were accessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. ResultsThe best-ranked references genes differed across the samples. GAPDH and PP2A were the most suitable for expression analysis in untreated tissues while GAPDH, α-TUB, and PP2A were ranked as the three most stably expressed genes in untreated roots, while NAC and CYP were the most stably expressed genes in stressed (i.e., treated) roots. The expression of UGPase, a key enzyme for CPP biosynthesis, was determined to further validate the selected RGs. ConclusionA total of 10 RGs can be used as reference genes of C. pilosula, however the appropriate one should be used as it may chance.

Abstract 4489: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1) ) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalo virus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and is a cosuppressor of AR in transient transfection assays. In this study, we report that the knockdown of ARID5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when ARID5a expression was down-regulated by siRNA technology. Androgen stimulates cell proliferation through activation of mammalian target of rapamycin complex 1 (mTORC1). However, the suppression of ARID5a does not affect androgen-activated mTOR activity. Furthermore, the expression of androgen-inducible and androgen-suppressive genes were not affected by suppression of ARID5a. We concluded that ARID5a is not a cosuppressor of AR. Meanwhile, flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of ARID5a expression. Oil Red O staining assays showed that the inhibition of ARID5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of ARID5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of ARID5a decreased the expression of cell survival / proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of ARID5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of ARID5a expression affected protein expression at translational and/or post-translational level. Our data suggested that knockdown of ARID5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of ARID5a expression suppressed the global protein synthesis. We showed that one of the four eIF2a kinases, general control non-derepressible 2 (GCN2) was activated by the suppression of ARID5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2017-4489

Identification and comparative analysis of the pearl oyster Pinctada fucata hemocytes microRNAs in response to Vibrio alginolyticus infection

MicroRNAs (miRNAs) are a class of noncoding RNA molecules that function as negative regulators of gene expression and play important roles in a wide spectrum of biological processes, including in immune response. However, the physiological regulation function of Pinctada fucata miRNAs, specially their immunomodulation has not been explored yet. Here, two small RNA libraries from hemocytes of P. fucata with or without Vibrio alginolyticus infection were constructed and sequenced using the high-throughput Illumina deep sequencing technology. In total, 11,939,992 and 11,083,327 raw reads, corresponding to 10,993,546 and 9,988,179 clean reads, were respectively obtained in the control and infected libraries. A total of 276 miRNAs, including 225 known miRNAs and 51 putative novel miRNAs, were identified by bioinformatic analysis. By using pairwise comparison between two libraries, 93 miRNAs were found to be significantly differentially expressed, with 42 and 51 miRNAs exhibiting up-regulation and down-regulation, respectively. Thereinto, some known miRNAs were considered to be immune-related. Real-time PCR were implemented for 6 miRNAs co-expressed in the control and infected samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. After miRNA targets were predicted, GO and KEGG pathway enrichment analysis were performed, and the results indicated that ten of the differentially expressed miRNAs were involved in immune-related pathways, and might participate in the host immune response to V. alginolyticus. These results of identification and comparative analysis of miRNAs might deepen our understanding of host-pathogen interactions and immune defense mechanisms in P. fucata.

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism

To map out key lipid-related pathways that lead to rapid triacylglyceride accumulation in oleaginous microalgae, RNA-Seq was performed with Tetraselmis sp. M8 at 24h after exhaustion of exogenous nitrogen to reveal molecular changes during early stationary phase. Further gene expression profiling by quantitative real-time PCR at 16–72h revealed a distinct shift in expression of the fatty acid/triacylglyceride biosynthesis and β-oxidation pathways, when cells transitioned from log-phase into early-stationary and stationary phase. Metabolic reconstruction modeling combined with real-time PCR and RNA-Seq gene expression data indicates that the increased lipid accumulation is a result of a decrease in lipid catabolism during the early-stationary phase combined with increased metabolic fluxes in lipid biosynthesis during the stationary phase. During these two stages, Tetraselmis shifts from reduced lipid consumption to active lipid production. This process appears to be independent from DGAT expression, a key gene for lipid accumulation in microalgae.