• Title/Summary/Keyword: quantitative real-time PCR (qPCR)

Search Result 244, Processing Time 0.022 seconds

Identification of relevant differential genes to the divergent development of pectoral muscle in ducks by transcriptomic analysis

  • Fan Li;Zongliang He;Yinglin Lu;Jing Zhou;Heng Cao;Xingyu Zhang;Hongjie Ji;Kunpeng Lv;Debing Yu;Minli Yu
    • Animal Bioscience
    • /
    • v.37 no.8
    • /
    • pp.1345-1354
    • /
    • 2024
  • Objective: The objective of this study was to identify candidate genes that play important roles in skeletal muscle development in ducks. Methods: In this study, we investigated the transcriptional sequencing of embryonic pectoral muscles from two specialized lines: Liancheng white ducks (female) and Cherry valley ducks (male) hybrid Line A (LCA) and Line C (LCC) ducks. In addition, prediction of target genes for the differentially expressed mRNAs was conducted and the enriched gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes signaling pathways were further analyzed. Finally, a protein-to-protein interaction network was analyzed by using the target genes to gain insights into their potential functional association. Results: A total of 1,428 differentially expressed genes (DEGs) with 762 being up-regulated genes and 666 being down-regulated genes in pectoral muscle of LCA and LCC ducks identified by RNA-seq (p<0.05). Meanwhile, 23 GO terms in the down-regulated genes and 75 GO terms in up-regulated genes were significantly enriched (p<0.05). Furthermore, the top 5 most enriched pathways were ECM-receptor interaction, fatty acid degradation, pyruvate degradation, PPAR signaling pathway, and glycolysis/gluconeogenesis. Finally, the candidate genes including integrin b3 (Itgb3), pyruvate kinase M1/2 (Pkm), insulin-like growth factor 1 (Igf1), glucose-6-phosphate isomerase (Gpi), GABA type A receptor-associated protein-like 1 (Gabarapl1), and thyroid hormone receptor beta (Thrb) showed the most expression difference, and then were selected to verification by quantitative real-time polymerase chain reaction (qRT-PCR). The result of qRT-PCR was consistent with that of transcriptome sequencing. Conclusion: This study provided information of molecular mechanisms underlying the developmental differences in skeletal muscles between specialized duck lines.

Morphological and genetic characterization and the nationwide distribution of the phototrophic dinoflagellate Scrippsiella lachrymosa in the Korean waters

  • Lee, Sung Yeon;Jeong, Hae Jin;You, Ji Hyun;Kim, So Jin
    • ALGAE
    • /
    • v.33 no.1
    • /
    • pp.21-35
    • /
    • 2018
  • The phototrophic dinoflagellate genus Scrippsiella is known to have a worldwide distribution. Here, we report for the first time, the occurrence of Scrippsiella lachrymosa in Korean waters. Unlike the other stains of S. lachrymosa whose cultures had been established from cysts in the sediments, the clonal culture of the Korean strain of S. lachrymosa was established from motile cells. When the sulcal plates of S. lachrymosa, which have not been fully described to date, were carefully examined using scanning electron microscopy, the Korean strain of S. lachrymosa clearly exhibited the anterior sulcal plate (s.a.), right sulcal plate (s.d.), left sulcal plate (s.s.), median sulcal plate (s.m.), and posterior sulcal plate (s.p.). When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of S. lachrymosa was ca. 1% different from those of two Norwegian strains of S. lachrymosa, the only strains for which LSU sequences have been reported. The internal transcribed spacer (ITS) rDNA sequence of the Korean strain of S. lachrymosa was also ca. 1% different from those of the Scottish and Chinese strains and 3% different from those of the Canadian, German, Greek, and Portuguese strains. Thus, the Korean S. lachrymosa strain has unique LSU and ITS sequences. The abundances of S. lachrymosa in the waters of 28 stations, located in the East, West, and South Sea of Korea, were quantified in four seasons from January 2016 to October 2017, using quantitative real-time polymerase chain reaction method and newly designed specific primer-probe sets. Its abundances were >$0.1cells\;mL^{-1}$ at eight stations in January and March 2016 and March 2017, and its highest abundance in Korean waters was $26cells\;mL^{-1}$. Thus, S. lachrymosa has a nationwide distribution in Korean waters as motile cells.

Short-Hairpin RNA-Mediated MTA2 Silencing Inhibits Human Breast Cancer Cell Line MDA-MB231 Proliferation and Metastasis

  • Lu, Jun;Jin, Mu-Lan
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.15 no.14
    • /
    • pp.5577-5582
    • /
    • 2014
  • Objective: To observe the effects of metastasis-associated tumor gene family 2 (MTA2) depletion on human breast cancer cell proliferation and metastasis. Methods: A short-hairpin RNA targeting MTA2 was chemically synthesized and transfected into a lentivirus to construct Lv-shMTA2 for infection into the MDA-MB231 human breast cancer cell line. At 48 hours after infection cells were harvested and mRNA and protein levels of MTA2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Cell viability and metastasis were assessed by CCK-8, wound-healing assay and Transwell assay, respectively. In addition, a xenograft model of human breast cancer was constructed to investigate cancerous cell growth and capacity for metastasis. Results: After infection with Lv-shMTA2, mRNA and protein levels of MTA2 was significantly reduced (p<0.05) and MDA-MB231 cell proliferation and metastasis were inhibited (p<0.05). In addition, mean tumor size was smaller than that in control group nude mice (p<0.05) and numbers of metastatic deposits in lung were lower than in control group mice (p<0.05). Depletion of MTA2 affected MMP-2 and apoptosis-related protein expression. Conclusions: For the first time to our knowledge we showed that MTA2 depletion could significantly inhibit human breast cancer cell growth and metastasis, implying that MTA2 might be involved in the progression of breast cancer. The role of MTA2 in breast cancer growth and metastasis might be linked with regulation of matrix metalloproteinase and apoptosis.

Effect of 17β-estradiol on Ecdysteroid Pathway Related Genes in the Brackish Water Flea Diaphanosoma celebensis (17β-estradiol이 기수산 물벼룩의 Ecdysteroid 경로에 미치는 영향)

  • In, Soyeon;Yoo, Jewon;Cho, Hayoung;Lee, Young-Mi
    • Journal of Marine Life Science
    • /
    • v.5 no.2
    • /
    • pp.35-42
    • /
    • 2020
  • 17β-estradiol (E2) is a natural hormone secreted by ovary, and continuously discharged from household and livestock wastewater into aquatic environment. Due to its strong estrogenic activity, it has adverse effects on development and reproduction in crustacean as an endocrine disrupting chemical. Although ecdysteroid signaling pathway play a key role in development in crustacean, little information on transcriptional modulation of ecdysteroid-related genes in response to E2 is available in small crustacean. Here, we investigated the acute toxicity of E2 to obtain 24-h LCx values in the brackish water flea Diaphanosoma celebensis. Time-dependent expression patterns of seven ecdysteroid pathway - related genes (CYP314a1, EcRA, EcRB, USP, ERR, Vtg, VtgR) were further examined using quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). As results, 24-h LC50 and LC10 values were 9.581 mg/l and 4.842 mg/l, respectively. The mRNA expression of CYP314a1, EcRA, USP, VtgR was significantly up-regulated at 12 or 24 h after exposure to E2. These findings indicate that E2 can affect their molting and reproduction by modulating the expression of ecdysteroid pathway - related in D. celebensis. This study will be useful for better understanding of molecular mode of action of endocrine disrupting chemicals on molting process in small crustacean.

microRNA Expression Profile in Patients with Stage II Colorectal Cancer: A Turkish Referral Center Study

  • Tanoglu, Alpaslan;Balta, Ahmet Ziya;Berber, Ufuk;Ozdemir, Yavuz;Emirzeoglu, Levent;Sayilir, Abdurrahim;Sucullu, Ilker
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.16 no.5
    • /
    • pp.1851-1855
    • /
    • 2015
  • Background: There are increasing data about microRNAs (miRNA) in the literature, providing abundant evidence that they play important roles in pathogenesis and development of colorectal cancer. In this study, we aimed to investigate the miRNA expression profiles in surgically resected specimens of patients with recurrent and non-recurrent colorectal cancer. Materials and Methods: The study population included 40 patients with stage II colorectal cancer (20 patients with recurrent tumors, and 20 sex and age matched patients without recurrence), who underwent curative colectomy between 2004 and 2011 without adjuvant therapy. Expression of 16 miRNAs (miRNA-9, 21, 30d, 31, 106a, 127, 133a, 133b, 135b, 143, 145, 155, 182, 200a, 200c, 362) was verified by quantitative real-time polymerase chain reaction (qRT-PCR) in all resected colon cancer tissue samples and in corresponding normal colonic tissues. Data analyses were carried out using SPSS 15 software. Values were statistically significantly changed in 40 cancer tissues when compared to the corresponding 40 normal colonic tissues (p<0.001). MiR-30d, miR-133a, miR-143, miR-145 and miR-362 expression was statistically significantly downregulated in 40 resected colorectal cancer tissue samples (p<0.001). When we compared subgroups, miRNA expression profiles of 20 recurrent cancer tissues were similar to all 40 cancer tissues. However in 20 non-recurrent cancer tissues, miR-133a expression was not significantly downregulated, moreover miR-133b expression was significantly upregulated (p<0.05). Conclusions: Our study revealed dysregulation of expression of ten miRNAs in Turkish colon cancer patients. These miRNAs may be used as potential biomarkers for early detection, screening and surveillance of colorectal cancer, with functional effects on tumor cell behavior.

Spatial-temporal distributions of the newly described mixotrophic dinoflagellate Gymnodinium smaydae in Korean coastal waters

  • Lee, Sung Yeon;Jeong, Hae Jin;Ok, Jin Hee;Kang, Hee Chang;You, Ji Hyun
    • ALGAE
    • /
    • v.35 no.3
    • /
    • pp.225-236
    • /
    • 2020
  • Gymnodinium smaydae is a newly described mixotrophic dinoflagellate that feeds on only Heterocapsa spp. and Scrippsiella acuminata among 19 tested algal prey. It is one of the fastest growing dinoflagellates when feeding, but does not grow well without prey. To investigate its spatial-temporal distributions in Korean waters, we quantified its abundance in water samples that were seasonally collected from 28 stations along the Korean Peninsula from April 2015 to October 2018, using quantitative real-time polymerase chain reactions. This dinoflagellate had a wide distribution, as reflected by the detection of G. smaydae cells at 23 of the sampling stations. However, this distribution had a strong seasonality; it was detected at 21 stations in the summer and only one station in winter. The abundance of G. smaydae was significantly and positively correlated with chlorophyll a concentration as well as with water temperature. However, there were no significant correlations between the abundance of G. smaydae and salinity, concentrations of nutrients, or dissolved oxygen concentration. During the study period, G. smaydae was present when water temperatures were 7.6-28.0℃, salinities were 9.6-34.1, concentrations of NO3 were not detectable-106.0 μM, and concentrations of PO4 were not detectable-3.4 μM. The highest abundance of G. smaydae was 18.5 cells mL-1 in the coastal waters of Jinhae in July 2017 when the chlorophyll a concentration was 127 mg m-3 and water temperature was 23.8℃. Therefore, the spatial-temporal distribution of G. smaydae in Korean coastal waters may be affected by chlorophyll a concentration and water temperature.

IL-6-miR-210 Suppresses Regulatory T Cell Function and Promotes Atrial Fibrosis by Targeting Foxp3

  • Chen, YingWei;Chang, GuoDong;Chen, XiaoJie;Li, YunPeng;Li, HaiYu;Cheng, Dong;Tang, Yi;Sang, HaiQiang
    • Molecules and Cells
    • /
    • v.43 no.5
    • /
    • pp.438-447
    • /
    • 2020
  • The aim of this study was to explore the role of IL-6-miR-210 in the regulation of Tregs function and atrial fibrosis in atrial fibrillation (AF). The levels of interleukin (IL)-6 and IL-10 in AF patients were detected by using ELISA. Proportions of Treg cells were detected by fluorescence activated cell sorting analysis in AF patients. The expression of Foxp3, α-SMA, collagen I and collagen III were determined by western blot. The atrial mechanocytes were authenticated by vimentin immunostaining. The expression of miR-210 was performed by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was used to predict potential targets of miR-210. The cardiomyocyte transverse sections in AF model group were observed by H&E staining. The myocardial filaments were observed by masson staining. The level of IL-6 was highly increased while the level of IL-10 (Tregs) was significantly decreased in AF patients as compared to normal control subjects, and IL-6 suppressed Tregs function and promoted the expression of α-SMA, collagen I and collagen III. Furthermore, miR-210 regulated Tregs function by targeting Foxp3, and IL-6 promoted expression of miR-210 via regulating hypoxia inducible factor-1α (HIF-1α). IL-6-miR-210 suppresses regulatory T cell function and promotes atrial fibrosis by targeting Foxp3.

Expression and Clinical Significance of MicroRNA-376a in Colorectal Cancer

  • Mo, Zhan-Hao;Wu, Xiao-Dong;Li, Shuo;Fei, Bing-Yuan;Zhang, Bin
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.15 no.21
    • /
    • pp.9523-9527
    • /
    • 2014
  • The incidence of colorectal cancer (CRC) is increasing in many Asian countries and microRNAs have already been proven to be associated with tumorigenesis. Currently, microRNA-376a (miR-376a) expression and association with clinical factors in CRC remains unclear. In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was carried out on 53 matched pairs of CRC and adjacent normal mucosa to investigate the expression levels of miR-376a. According to the high or low expression of miR-376a, patients were divided into two groups. The relationship between miR-376a expression and clinicopathological factors of 53 patients was evaluated. Survival analysis of 53 CRC patients was performed with clinical follow-up information and survival curves were assessed by the Kaplan-Meier method. Immunohistochemistry (IHC) staining was performed on sections of paraffin-embedded tissue to investigate the vascular endothelial growth factor (VEGF) expression. MiR-376a showed low expression in cancer tissues compared to the adjacent normal tissues and altered high miR-376a expression tended to be positively correlated with advanced lymph node metastasis and shorter patient survival. VEGF IHC positivity was significantly more common in patients with high expression levels of miR-376a.Those results demonstrated that miR-376a may be a meaningful prognostic biomarker and potential therapeutic target in colorectal cancer.

Silencing of Rac3 Inhibits Proliferation and Induces Apoptosis of Human Lung Cancer Cells

  • Liu, Tie-Qin;Wang, Ge-Bang;Li, Zheng-Jun;Tong, Xiang-Dong;Liu, Hong-Xu
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.16 no.7
    • /
    • pp.3061-3065
    • /
    • 2015
  • Background: Rac3, a member of the Rac family of small guanosine triphosphatases (GTPases), regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. Overexpression of Rac3 has been reported in several human cancers. However, the role of Rac3 in lung cancer (LC) has not been determined in detail. The purpose of this study was to investigate the effect of silencing of Rac3 expression in human LC cells and the consequences for cell survival. Materials and Methods: Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down the Rac3 gene. Gene and protein expression was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. LC cell apoptosis was examined by annexin V-APC /propidium iodide staining. Results: Efficient silencing of Rac3 strongly inhibited A549 cell proliferation and colony formation ability, and significantly decreased tumor growth. Moreover, flow cytometry analysis showed that knockdown of Rac3 led to G2/M phase cell cycle arrest as well as an excess accumulation of cells in the G1 and S phase. Conclusions: Thus, functional analysis using shRNAs revealed a critical role for Rac3 in the tumor growth of LC cells. shRNA silencing of Rac3 could provide an effective strategy to treat LC.

Expression and Significance of Microsomal Prostaglandin Synthase-1 (mPGES-1) and Beclin-1 in the Development of Prostate Cancer

  • Xu, Lu-Wei;Qian, Ming;Jia, Rui-Peng;Xu, Zheng;Wu, Jian-Ping;Li, Wen-Cheng;Huang, Wen-Bin;Chen, Xing-Guo
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.13 no.4
    • /
    • pp.1639-1644
    • /
    • 2012
  • The aim of this study was to investigate the expression and significance of microsomal prostaglandin synthase-1 (mPGES-1) and Beclin-1 in the development of prostate cancer (PCa). Immunohistochemistry was performed on paraffin-embedded sections with rabbit polyclonal against mPGES-1 and Beclin-1 in 40 PCa, 40 benign prostatic hyperplasia (BPH) and 10 normal prostate specimens for this purpose. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied for mRNA expression of mPGES-1 and Beclin-1, while MTT assays were used to ascertain the best working concentration of the mPGES-1 inhibitor (CAY10526). The effect of CAY10526 treatment on expression of Beclin-1 in DU-145 cells was studied using Western blot analysis. Localization of Beclin-1 and mPGES-1 was in endochylema. Significant differences in expression was noted among PCa, BPH and normal issues (P<0.05). Beclin-1 expression inversely correlated with mPGES-1 expression in PCa tissue (P<0.05). CAY10526 could significantly block mPGES-1 expression and the proliferation of DU-145 cells (P<0.05), while increasing Beclin-1 levels (P<0.05). Overexpression of mPGES-1 could decrease the autophagic PCa cell death. Inhibiting the expression of mPGES-1 may lead to DU-145 cell death and up-regulation of Beclin-1. The results suggest that inhibition of mPGES-1 may have therapeutic potential for PCa in the future.