scholarly journals A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Man Wang ◽  
Feng Zhou ◽  
Hong Mei Wang ◽  
De Xing Xue ◽  
Yao-Guang Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Posttranscriptional Regulation ◽  
Expression Patterns ◽  
Critical Role ◽  
Temperature Acclimation ◽  
Northern Blotting ◽  
Protein Levels ◽  
Molecular Functions

Abstract Background Plant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined. Results Here an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14. Conclusions These data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

scholarly journals Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns

2012 ◽  
Vol 7 (5) ◽  
pp. 829-838 ◽  
Author(s):  
Veronica Sanchez-Freire ◽  
Antje D Ebert ◽  
Tomer Kalisky ◽  
Stephen R Quake ◽  
Joseph C Wu
Keyword(s):  
Gene Expression ◽  
Comparative Analysis ◽  
Single Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Patterns ◽  
Gene Expression Patterns

Mammaglobin B expression in human endometrial cancer

2008 ◽  
Vol 18 (5) ◽  
pp. 1090-1096 ◽  
Author(s):  
R. A. Tassi ◽  
E. Bignotti ◽  
M. Falchetti ◽  
S. Calza ◽  
A. Ravaggi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endometrial Cancer ◽  
Real Time ◽  
Real Time Pcr ◽  
Messenger Rna ◽  
Endometrial Cells ◽  
Protein Levels ◽  
Fresh Frozen ◽  
Endometrioid Endometrial Cancer ◽  
Polymerase Chain

Mammaglobin B (MGB-2) is an uteroglobin gene family member recently found highly differentially expressed in ovarian cancer by gene expression profiling. To evaluate its potential as a novel endometrial cancer biomarker, in this study we quantified and compared MGB-2 expression at messenger RNA and protein levels in endometrial tumors (endometrioid endometrial cancer [EEC]) with different grades of differentiation. MGB-2 expression was evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) in fresh frozen biopsies and paraffin-embedded tissues derived from a total of 70 patients including 50 primary EEC and 20 normal endometria (NECs). High levels of MGB-2 gene expression were detected in 10 of 11 EEC G1 cases (91%), 16 of 17 EEC G2 cases (94%), and 6 of 22 EEC G3 cases (27%) by real-time PCR. In contrast, normal endometrial cells expressed low to negligible levels of MGB-2 by real-time PCR (P= 0.002 EEC vs NEC). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (P< 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were also statistically significant (G2 vs G3 P< 0.001, G1 vs G3 P= 0.016). MGB-2 protein expression was detected in 31 (86%) of 36 EEC and 0 of 5 atrophic NEC controls, while seven of eight (88%) of the proliferative/secretory/hyperplastic NECs focally expressed MGB-2 by IHC. MGB-2 is highly expressed in EEC, particularly in well- and moderately differentiated tumors, and may represent a novel molecular marker for EEC.

scholarly journals Gene expression patterns in bone following lipopolysaccharide stimulation

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
Jing Yang ◽  
Nan Su ◽  
Xiaolan Du ◽  
Lin Chen
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Real Time ◽  
Real Time Pcr ◽  
Early Stage ◽  
System Development ◽  
Expression Patterns ◽  
Osteoclast Differentiation ◽  
Gene Expression Patterns ◽  
Expression Levels

AbstractBone displays suppressed osteogenesis in inflammatory diseases such as sepsis and rheumatoid arthritis. However, the underlying mechanisms have not yet been clearly explained. To identify the gene expression patterns in the bone, we performed Affymetrix Mouse Genome 430 2.0 Array with RNA isolated from mouse femurs 4 h after lipopolysaccharide (LPS) administration. The gene expressions were confirmed with real-time PCR. The serum concentration of the N-terminal propeptide of type I collagen (PINP), a bone-formation marker, was determined using ELISA. A total of 1003 transcripts were upregulated and 159 transcripts were downregulated (more than twofold upregulation or downregulation). Increased expression levels of the inflammation-related genes interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were confirmed from in the period 4 h to 72 h after LPS administration using real-time PCR. Gene ontogene analysis found four bone-related categories involved in four biological processes: system development, osteoclast differentiation, ossification and bone development. These processes involved 25 upregulated genes. In the KEGG database, we further analyzed the transforming growth factor β (TGF-β) pathway, which is strongly related to osteogenesis. The upregulated bone morphogenetic protein 2 (BMP2) and downregulated inhibitor of DNA binding 4 (Id4) expressions were further confirmed by real-time PCR after LPS stimulation. The osteoblast function was determined through examination of the expression levels of core binding factor 1 (Cbfa1) and osteocalcin (OC) in bone tissues and serum PINP from 4 h to 72 h after LPS administration. The expressions of OC and Cbfa1 decreased 6 h after administration (p < 0.05). Significantly suppressed PINP levels were observed in the later stage (from 8 h to 72 h, p < 0.05) but not in the early stage (4 h or 6 h, p > 0.05) of LPS stimulation. The results of this study suggest that LPS induces elevated expressions of skeletal system development- and osteoclast differentiation-related genes and inflammation genes at an early stage in the bone. The perturbed functions of these two groups of genes may lead to a faint change in osteogenesis at an early stage of LPS stimulation. Suppressed bone formation was found at later stages in response to LPS stimulation.

103 GENE EXPRESSION PROFILES OF VITRIFIED MOUSE EMBRYOS DETECTED BY MICROARRAYS

2006 ◽  
Vol 18 (2) ◽  
pp. 160
Author(s):  
S. Mamo ◽  
Sz. Bodo ◽  
Z. Polgar ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Differentially Expressed ◽  
Analysis Tool ◽  
Cell Stage ◽  
Base Medium ◽  
Independent Analysis

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 �g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes. This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002).

scholarly journals Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaowei Wang ◽  
Zhijun Wu ◽  
Wenqi Bao ◽  
Hongyan Hu ◽  
Mo Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Stress Responses ◽  
Real Time Pcr ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Expression Patterns ◽  
Polygonum Cuspidatum ◽  
Quantitative Real Time Pcr ◽  
Different Tissues

Abstract Background Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. Results Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. Conclusions We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.

Gene expression patterns for GDNF and its receptors in the human putamen affected by Parkinson's disease: A real-time PCR study

2006 ◽  
Vol 252 (1-2) ◽  
pp. 160-166 ◽  
Author(s):  
Cristina M. Bäckman ◽  
Lufei Shan ◽  
Ya Jun Zhang ◽  
Barry J. Hoffer ◽  
Sherry Leonard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Patterns ◽  
Gene Expression Patterns

scholarly journals Expression patterns of HMGA2 in the placenta during pregnancy

2020 ◽  
Author(s):  
Lars Burchardt ◽  
Andrea Gottlieb ◽  
Burkhard M. Helmke ◽  
Werner Wosniok ◽  
Wolfgang Kuepker ◽  
...  
Keyword(s):  
Real Time ◽  
Gestational Age ◽  
Real Time Pcr ◽  
Expression Patterns ◽  
Critical Role ◽  
First Trimester ◽  
Postnatal Life ◽  
Invasive Growth ◽  
Quantitative Real Time Pcr ◽  
Mesenchymal Transition

AbstractBackgroundHigh-mobility group AT-hook 2 (HMGA2) expression can be detected in many embryonic and fetal tissues but becomes down-regulated during postnatal life except for many benign and malignant tumors. In the latter case, its expression has been correlated with epithelial-mesenchymal transition and invasive growth. The placenta contributes essentially to proper development of the embryo and the fetus. In a tumor-like manner it shows rapid invasive growth during the first weeks of gestation. To address the possible role of HMGA2 during placental development, we have measured its expression throughout the prenatal period and in term placentae by mRNA quantification as well as by immunohistochemistry.MethodsExpression of HMGA2 and HPRT was measured on 89 fetal placentas, encompassing calendar gestational age of five to 41 weeks, using quantitative real time-PCR. In eleven cases in addition immunohistochemistry was used to determine the localization of HMGA2 and to compare with data obtained by quantitative real time-PCR.ResultsThe expression of HMGA2 was found to be inversely correlated with gestational age (p < 0.001). For the first part of the first trimester the level of HMGA2 is high. After that the expression shows a decline down to a baseline level where it remains until birth. HMGA2 protein was mainly detected in the nuclei of the stromal cells in the placental villi.ConclusionsDuring pregnancy, the expression of HMGA2 follows a non-linear pattern of decrease. In the first trimester, from two to three weeks after the implantation of the conceptus until the blood supply is established (hypoxic phase), the expression is high, indicating a critical role during early development and in the control of its invasive behavior, respectively.

scholarly journals Intestinal dysmotility after bowel resection in rats is associated with decreased ghrelin and vimentin expression and loss of intestinal cells of Cajal

2020 ◽  
Author(s):  
Igor Sukhotnik ◽  
Yoav Ben-Shahar ◽  
Yulia Pollak ◽  
Shlomi Cohen ◽  
Hadar Moran-Lev ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Motility ◽  
Bowel Resection ◽  
Down Regulation ◽  
Intestinal Transit Time ◽  
Protein Levels ◽  
Cells Of Cajal

The purpose of this study was to evaluate the mechanisms of intestinal motility in a rat model of short bowel syndrome (SBS). Rats were divided into three groups: Sham rats underwent bowel transection; SBS-NSI rats underwent a 75% bowel resection and presented with normal intestinal size (NSI) at sacrifice and hypermotility patterns; SBS-DYS showed dysmotile (DYS) enlarged intestine and inhibited motility patterns. Animals were sacrificed after 2 weeks. Illumina's Digital Gene Expression (DGE) analysis was used to determine the intestinal motility-related gene expression profiling in mucosal samples. Intestinal motility-related and interstitial cells of Cajal (ICC) genes and protein expression in intestinal muscle layer were determined using Real Time PCR, Western blotting and immunohistochemistry. Gastrointestinal tract motility was studied by microcomputer tomography. From ten Ca2+ signaling pathway related genes, six genes in jejunum and seven genes in ileum were down-regulated in SBS vs Sham animals. Down regulation of TMEM16A mRNA and protein was confirmed by Real Time PCR. Rapid intestinal transit time in SBS-NSI rats correlated with mild decrease in TMEM 16A, c-kit and vimentin mRNA and protein expression (vs Sham animals). SBS-DYS rats demonstrated enlarged intestinal loops and delayed small intestinal emptying (on imaging studies) that were correlated with marked down-regulation in TMEM 16A, c-kit, vimentin, ghrelin mRNA and protein levels compared to the other two groups. In conclusion, two weeks following massive bowel resection in rats, impaired intestinal motility was associated with decreased vimentin and ghrelin gene and protein levels as well as loss of ICC (c-kit and TMEM16A).

scholarly journals Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Toshio Hattori ◽  
Naomi Ogura ◽  
Miwa Akutsu ◽  
Mutsumi Kawashima ◽  
Suguru Watanabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Temporomandibular Joint ◽  
Real Time ◽  
Real Time Pcr ◽  
Protein Production ◽  
Critical Role ◽  
Synovial Fibroblasts ◽  
Temporomandibular Joint Disorders ◽  
Pcr Analysis ◽  
Dependent Manner

Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD.

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