scholarly journals Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle

2004 ◽  
Vol 32 (2) ◽  
pp. 449-466 ◽  
Author(s):  
S Bauersachs ◽  
S Rehfeld ◽  
SE Ulbrich ◽  
S Mallok ◽  
K Prelle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Expression Profiles ◽  
Cdna Array ◽  
Oestrous Cycle ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Expression Levels ◽  
Starting Point ◽  
Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

scholarly journals 230TRANSCRIPTOMICS ANALYSIS OF CHANGES IN GENE EXPRESSION IN BOVINE OVIDUCT EPITHELIAL CELLS DURING THE ESTROUS CYCLE

2004 ◽  
Vol 16 (2) ◽  
pp. 236
Author(s):  
S. Bauersachs ◽  
S. Rehfeld ◽  
S. Koelle ◽  
S. Mallok ◽  
K. Prelle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Estrous Cycle ◽  
Cdna Array ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Ciliated Cells ◽  
Expression Levels ◽  
Functional Changes ◽  
Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs;; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n=42) were expressed at a higher level at estrus;; for the other (n=42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell–cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

Identification of Angiogenesis/Metastases Genes Predicting Chemoradiotherapy Response in Patients With Laryngopharyngeal Carcinoma

2007 ◽  
Vol 25 (11) ◽  
pp. 1369-1376 ◽  
Author(s):  
Ian Ganly ◽  
Simon Talbot ◽  
Diane Carlson ◽  
Agnes Viale ◽  
Ellie Maghami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cdna Microarray ◽  
Expression Profiles ◽  
External Validation ◽  
Cdna Array ◽  
Disease Free Survival ◽  
Independent Set ◽  
Rt Pcr ◽  
Locoregional Failure

Purpose To identify genes related to angiogenesis/metastasis that predict locoregional failure in patients with laryngopharyngeal cancer (LPC) undergoing chemoradiotherapy (CRT) treatment. Methods Tumor tissue was collected and snap-frozen from 35 sequential patients with histologically confirmed LPC being treated with CRT. Gene expression analysis was performed using a novel cDNA array consisting of 277 genes functionally associated with angiogenesis (n = 152) and/or metastasis (n = 125). Locoregional response was correlated to the gene expression profiles to identify genes associated with outcome. These genes were internally validated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and validated externally by immunohistochemistry analysis on an independent set of patients. Results Locoregional failure occurred in nine of 35 patients. Seventeen genes from the cDNA microarray correlated with locoregional failure (two-sample t test, P < .05). Seven genes were chosen for additional analysis based on the availability of antibodies for immunohistochemistry. Of these seven genes, real-time RT-PCR validated four genes: MDM2, VCAM-1, erbB2, and H-ras (Wilcoxon rank sum test, P = .008, .02, .04, and .04, respectively). External validation by immunohistochemistry confirmed MDM2 and erbB2 as being predictive of locoregional response. Controlling for stage of disease, positivity for MDM2 or erbB2 was an independent negative predictor of locoregional disease-free survival. Conclusion Genomic screening by cDNA microarray and validation internally by real-time RT-PCR and externally by immunohistochemistry have identified two genes (MDM2 and erbB2) as predictors of locoregional failure in LPC patients treated with CRT. The role of these genes in treatment selection and the functional basis for their activity in CRT response merit additional consideration.

scholarly journals Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR

2021 ◽  
Vol 22 (17) ◽  
pp. 9349
Author(s):  
Nicole Rachinger ◽  
Stefan Fischer ◽  
Ines Böhme ◽  
Lisa Linck-Paulus ◽  
Silke Kuphal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Expression Profiles ◽  
Cdna Array ◽  
Gene Expression Profiles ◽  
Strong Impact ◽  
Rna Seq ◽  
Rt Pcr ◽  
Gene Information ◽  
Insight Into

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

265 DICER GENE EXPRESSION IN PRE-IMPLANTATION MOUSE EMBRYOS: IMPLICATION OF TRANSCRIPTION REGULATION AT THE BLASTOCYST STAGE

2007 ◽  
Vol 19 (1) ◽  
pp. 249
Author(s):  
X. S. Cui ◽  
X. H. Shen ◽  
X. Y. Li ◽  
J. M. Kim ◽  
N. H. Kim
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Transcription Factors ◽  
Real Time ◽  
Expression Profiles ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Expression Levels ◽  
Mouse Oocytes ◽  
Stage Embryo

Dicer is an RNAse III enzyme that is related to the generation of the microRNAs involved in the gene silencing pathway. In order to obtain insight into the role of Dicer in early embryo development, we first evaluated its gene expression levels in mouse oocytes and embryos during in vitro development. The relative abundance of Dicer1 transcripts was established by real-time RT-PCR using the 2-ddCt method. H2a was applied as an internal standard to normalize the real-time RT-PCR reaction efficiency and quantify Dicer1 mRNA. Relatively high expression levels of mRNA in germinal vesicle-stage oocytes steadily decreased up to the 2-cell stage embryo, and then expression remained during morulae and blastocyst formation. Protein synthesis of Dicer was also observed in the mouse oocytes and early embryos. Specific silencing of mRNA expression and protein synthesis by RNA interference (siRNA) did not inhibit developmental events up to the blastocyst (BL) stage. However, Dicer1 siRNA reduced (P &lt;0.05) total nuclei numbers in the BL-stage embryos (Dicer1: 77.2�4.2 vs. control: 62.7�3.1). Real-time RT-PCR also confirmed that, following Dicer1 siRNA microinjection into zygotes, transcription levels of several non-target genes, Cdc42, Cdh1, Dbc2, ILK, Tuba1, Plat, and Tie1, were not changed in blastocyst-stage embryos. However, selected transcription factors, Pou5f1 (P &lt;0.01), Nanog (P &lt;0.005), and Sox2 (P &lt;0.01), in blastocysts were significantly down-regulated. Additionally, POU5F1 protein synthesis was also reduced. Using Applied Biosystem microarray technology, we compared gene expression profiles in control and Dicer1 siRNA microinjected blastocysts. This technique confirmed that 397 or 737 of 16354 genes were up- or down-regulated, respectively, following siRNA microinjection (P &lt;0.05), including 52 transcription factors. The results suggest that expression of Dicer regulates gene expression at the blastocyst-stage embryo for cell fate, possibly by the transcription control.

scholarly journals Identification of key genes in invasive clinically non-functioning pituitary adenoma by integrating analysis of DNA methylation and mRNA expression profiles

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Sen Cheng ◽  
Weiyan Xie ◽  
Yazhou Miao ◽  
Jing Guo ◽  
Jichao Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Pituitary Adenoma ◽  
Candidate Genes ◽  
Tumor Invasion ◽  
Expression Profiles ◽  
Methylation Status ◽  
Rt Pcr ◽  
Expression Levels ◽  
Key Genes

Abstract Background Tumor surrounding the internal carotid artery or invading to the cavernous sinus is an important characteristic of invasive pituitary adenoma, and a pivotal factor of tumor residue and regrowth. Without specific changes in serum hormone related to the adenohypophyseal cell of origin, clinically non-functioning pituitary adenoma is more likely to be diagnosed at invasive stages compared with functioning pituitary adenoma. The underlying mechanism of tumor invasion remains unknown. In this study, we aimed to identify key genes in tumor invasion by integrating analyses of DNA methylation and gene expression profiles. Method Genome-wide DNA methylation and mRNA microarray analysis were performed for tumor samples from 68 patients at the Beijing Tiantan Hospital. Differentially expressed genes and methylated probes were identified based on an invasive vs non-invasive grouping. Differentially methylated probes in the promoter region of targeted genes were assessed. Pearson correlation analysis was used to identify genes with a strong association between DNA methylation status and expression levels. Pyrosequencing and RT-PCR were used to validate the methylation status and expression levels of candidate genes, respectively. Results A total of 8842 differentially methylated probes, located on 4582 genes, and 661 differentially expressed genes were identified. Both promoter methylation and expression alterations were observed for 115 genes with 58 genes showing a negative correlation between DNA methylation status and expression level. Nineteen genes that exhibited notably negative correlations between DNA methylation and gene expression levels, are involved in various gene ontologies and pathways, or played an important role in different diseases, were regarded as candidate genes. We found an increased methylation with a decreased expression of PHYHD1, LTBR, C22orf42, PRR5, ANKDD1A, RAB13, CAMKV, KIFC3, WNT4 and STAT6, and a decreased methylation with an increased expression of MYBPHL. The methylation status and expression levels of these genes were validated by pyrosequencing and RT-PCR. Conclusions The DNA methylation and expression levels of PHYHD1, LTBR, MYBPHL, C22orf42, PRR5, ANKDD1A, RAB13, CAMKV, KIFC3, WNT4 and STAT6 are associated with tumor invasion, and these genes may become the potential genes for targeted therapy.

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

2004 ◽  
Vol 287 (4) ◽  
pp. G875-G885 ◽  
Author(s):  
Carine Strup-Perrot ◽  
Denis Mathé ◽  
Christine Linard ◽  
Dominique Violot ◽  
Fabien Milliat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Expression Profiles ◽  
Cdna Array ◽  
Muscularis Propria ◽  
Gelatin Zymography ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Radiation Enteritis ◽  
Mrna Levels ◽  
Fixed Tissue

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and imunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland

1996 ◽  
Vol 16 (1) ◽  
pp. 27-37 ◽  
Author(s):  
L Gabou ◽  
M Boisnard ◽  
I Gourdou ◽  
H Jammes ◽  
J-P Dulor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Untranslated Regions ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Prolactin Gene ◽  
New Zealand White Rabbits ◽  
Rna Probe

ABSTRACT cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5′ and 3′ untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93–78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

scholarly journals Analysis of Expressed Sequence Tag Data and Gene Expression Profiles Involved in Conidial Germination of Fusarium oxysporum

2006 ◽  
Vol 72 (2) ◽  
pp. 1667-1671 ◽  
Author(s):  
Ye Deng ◽  
Haitao Dong ◽  
Qingchao Jin ◽  
Cheng'en Dai ◽  
Yongqi Fang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Fusarium Oxysporum ◽  
Cdna Library ◽  
Expressed Sequence Tag ◽  
Expression Profiles ◽  
Cdna Array ◽  
Gene Expression Profiles ◽  
Conidial Germination ◽  
Expressed Sequence

ABSTRACT We obtained 3,372 tentative unique transcripts (TUTs) from a cDNA library of Fusarium oxysporum. A cDNA array with 3,158 TUTs was produced to analyze gene expression profiles in conidial germination. It seems that ras and other signaling genes, e.g., ccg, cooperatively initiate conidial germination in Fusarium by increasing protein synthesis.

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