DNA Methylation Inhibitor 5-Aza-2′-Deoxycytidine Modulates Endometrial Receptivity Through Upregulating HOXA10 Expression

2018 ◽  
Vol 26 (6) ◽  
pp. 839-846 ◽  
Author(s):  
Li Wang ◽  
Ya Jing Tan ◽  
Min Wang ◽  
Yi Fei Chen ◽  
Xin Yan Li
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
Messenger Rna ◽  
Dna Methyltransferase ◽  
Target Genes ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Integrin Subunit ◽  
Chain Reaction ◽  
Polymerase Chain

Endometrial receptivity is a critical factor for embryo implantation. A decrease in endometrial homeobox A10 (HOXA10) expression is associated with hypermethylation of its promoter and lower endometrial receptivity in animals and humans. 5-Aza-2′-deoxycytidine (AZA) is a DNA methyltransferase inhibitor. However, whether demethylation of the HOXA10 gene could increase the receptivity of the human endometrium remains unknown. Homeobox A10 promoter methylation was analyzed using bisulfite genomic sequencing polymerase chain reaction. Quantitative real time polymerase chain reaction and Western blotting were used to analyze the expression of HOXA10 and its downstream target genes (integrin subunit β 3 [ITGB3] and insulin growth factor binding protein 1 [IGFBP1]) in Ishikawa cells treated with or without AZA for 24 hours. Their protein expression was analyzed with or without HOXA10 siRNA treatment. The effect of AZA on embryo implantation was examined using a Jeg-3 spheroid-endometrial cell attachment assay. The percentage of methylated CpG islands in the HOXA10 promoter was 72.0% without AZA treatment. However, it was 38% and 35% in the 1 and 10 μM AZA treatment groups, respectively. 5-Aza-2′-deoxycytidine strongly induced the expression of HOXA10, ITGB3, and IGFBP1 messenger RNA and their protein expression. Homeobox A10 knockdown led to decreased expression of HOXA10, ITGB3, and IGFBP1, with or without AZA treatment. The attachment rate of Jeg-3 spheroids increased significantly from 82% (control) to 95% (AZA 1 μM) and 96% (AZA 10 μM) after AZA treatment. 5-Aza-2′-deoxycytidine could upregulate the expression of ITGB3 and IGFBP1 via HOXA10 upregulation, and upregulation of ITGB3 and IGFBP1 plays an important role in endometrial receptivity during implantation. 5-Aza-2′-deoxycytidine may improve endometrial receptivity by upregulating the expression of HOXA10.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

Novel Method for Simultaneous Analysis of p53 and K-ras Mutations and p53 Protein Expression in Single Histologic Sections

2001 ◽  
Vol 125 (3) ◽  
pp. 347-352
Author(s):  
Kazuya Yamashita ◽  
Tsutomu Yoshida ◽  
Hiroshi Shinoda ◽  
Isao Okayasu
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
P53 Protein ◽  
Single Strand Conformation Polymorphism ◽  
Antigen Retrieval ◽  
Cancer Tissue ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Novel Method ◽  
Polymerase Chain

Abstract Background and Objective.—Abnormal protein expression and gene mutation should be examined on exactly identified lesions. To perform simultaneous analyses of oncogene or tumor suppressor gene mutations and related protein expression in single histologic sections, we have developed a novel method using an antigen-retrieval solution for a polymerase chain reaction template before immunohistochemical staining. Methods.—Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-μm-thick, formalin-fixed, paraffin-embedded histologic sections at 96°C for 20 minutes. Polymerase chain reaction–single-strand conformation polymorphism analysis was conducted for p53 (exons 5 through 9) and K-ras (exons 1 and 2), and the histologic sections were then immunostained with monoclonal antibody against p53. Results.—DNA analysis of antigen-retrieval solutions was possible in all 20 cases and revealed completely consistent results (100%) with fresh cancer tissue and microdissected cancer tissue of paraffin-embedded histologic sections. With this method, K-ras mutations were positive in 10 of 20 cases (exon 1 in 9 cases and exon 2 in 1 case) and p53 mutations were positive in 9 of 20 cases (exon 5 in 4 cases, exon 6 in 1, exon 7 in 3, and exon 8 in 1 case), with 8 of the 9 p53 mutation cases showing diffuse p53 protein expression on immunostaining. Base alterations of all abnormal conformers were confirmed with direct sequencing. For polymerase chain reaction–single-strand conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was found to be the optimal antigen-retrieval solution. Conclusions.—This newly developed method can be used for routine immunostaining and genetic analysis with single histologic sections.

scholarly journals Expression of DROSHA and DICER genes in peripheral blood leukocytes in lung sarcoidosis

2018 ◽  
Vol 90 (3) ◽  
pp. 21-24
Author(s):  
I E Malysheva ◽  
O V Balan ◽  
E L Tikhonovich ◽  
T O Volkova
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Peripheral Blood ◽  
Messenger Rna ◽  
Peripheral Blood Leukocytes ◽  
Chain Reaction ◽  
Blood Leukocytes ◽  
Healthy Donors ◽  
Polymerase Chain

Aim. To study the expression level of the genes DROSHA and DICER in peripheral blood leukocytes (PBL) of patients with sarcoidosis of the lungs Materials and methods. The study included 32 patients diagnosed with persistent lung sarcoidosis (mean age 41.56±1.27 years) and 36 healthy donors (control; mean age 42.79±1.95 years). The level of expression of messenger RNA (mRNA) of the genes DROSHA and DICER were determined in PBL of healthy donors and patients with sarcoidosis of the lung by polymerase chain reaction in real time. Results. As a result of the conducted researches it is established that the level of drosha gene expression in PBL patients with sarcoidosis of lungs is significantly reduced in comparison with the control (p

scholarly journals A beginner’s guide to RT-PCR, qPCR and RT-qPCR

2020 ◽  
Vol 42 (3) ◽  
pp. 48-53 ◽  
Author(s):  
Grace Adams
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
Life Science ◽  
Science Research ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mrna Quantitation ◽  
Polymerase Chain

The development of the polymerase chain reaction (PCR), for which Kary Mullis received the 1992 Novel Prize in Chemistry, revolutionized molecular biology. At around the time that prize was awarded, research was being carried out by Russel Higuchi which led to the discovery that PCR can be monitored using fluorescent probes, facilitating quantitative real-time PCR (qPCR). In addition, the earlier discovery of reverse transcriptase (in 1970) laid the groundwork for the development of RT-PCR (used in molecular cloning). The latter can be coupled to qPCR, termed RT-qPCR, allowing analysis of gene expression through messenger RNA (mRNA) quantitation. These techniques and their applications have transformed life science research and clinical diagnosis.

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