Preparation of biomimetic gene hydrogel via polymerase chain reaction for cell-free protein expression

2019 ◽  
Vol 63 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Feng Li ◽  
Wenting Yu ◽  
Xue Zhang ◽  
Xiaocui Guo ◽  
Xihan Xu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
Chain Reaction ◽  
Free Protein ◽  
Polymerase Chain

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 The Study of HLA-G Gene and Protein Expression in Patients withRecurrent Miscarriage

2019 ◽  
Vol 9 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Elnaz Mosaferi ◽  
Nazila Alizadeh Gharamaleki ◽  
Laya Farzadi ◽  
Jafar Majidi ◽  
Zohreh Babaloo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
Sample Size ◽  
G Protein ◽  
Recurrent Miscarriage ◽  
Chain Reaction ◽  
Gene And Protein Expression ◽  
Protein Expressions ◽  
Polymerase Chain

Purpose: Although it has been frequently confirmed that HLA-G plays an important role in thereproduction and pregnancy, the pattern of HLA-G gene and its protein expression are rarelyaddressed in studies. Therefore we conducted this study in regard to evaluate the HLA-G geneand its protein expression in the women’s placenta with recurrent miscarriage.Methods: Placental samples were obtained from the women who were admitted for deliveryor abortion in Al Zahra and Taleghani hospitals, Tabriz, Iran. HLA-G gene expression wasdetermined by real-time polymerase chain reaction (PCR) and HLA-G protein expression wasassessed by western blotting and immunofluorescence staining in the tissue samples.Results: The results showed a significant decrease in the expression of gene and proteins ofHLA-G in the women with recurrent miscarriage compared to the control placental tissues.Conclusion: According to the obtained results, it was concluded that the decrement of HLA-Ggene and protein expressions are associated with recurrent miscarriage. Since there areconflicting results from other studies, it is suggested to conduct a more comprehensive similarstudy with greater sample size.

Expression of the Serine Protease Kallikrein 7 and Its Inhibitor Antileukoprotease Is Decreased in Prostate Cancer

2008 ◽  
Vol 132 (11) ◽  
pp. 1796-1801 ◽  
Author(s):  
Qiang Xuan ◽  
Xiaoli Yang ◽  
Linjian Mo ◽  
Fengyu Huang ◽  
Youhong Pang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Western Blot ◽  
Chain Reaction ◽  
Prostate Epithelial Cells ◽  
Polymerase Chain ◽  
Prostate Cancers ◽  
Benign Prostate

Abstract Context.—Kallikreins are a subgroup of serine proteases with diverse physiologic functions. It has been confirmed that kallikrein 7 (KLK7) is differentially expressed in ovarian and breast cancer. Antileukoprotease (ALP) has been shown to be a specific inhibitor of human kallikrein 7 (hK7). Antileukoprotease overexpression is commonly associated with aggressive, high-risk, or metastatic cancer originating from various organs. Objective.—To investigate the expression and potential role of hK7 and its inhibitor ALP in prostate cancer. Design.—The mRNA expression of KLK7 and ALP transcript in benign prostate epithelial cells and prostate cancers was evaluated by semiquantitative reverse transcription–polymerase chain reaction. We examined hK7 and ALP protein expression by immunohistochemistry in 20 normal prostate tissues, 50 benign prostatic hyperplasia tissues, and 103 prostate cancers. Western blot examination showed protein expression of hK7 and ALP in benign prostate epithelial cells and prostate cancer cell lines. Results.—Semiquantitative polymerase chain reaction examination revealed that the mRNA level of KLK7 and ALP was significantly decreased in prostate cancers compared with that in benign prostate epithelial cells (P < .001). Immunohistochemical expression of hK7 was observed in prostate epithelial cells, whereas little or no staining was observed in prostate cancer. Western blot analysis revealed that hK7 and ALP were decreased in malignant prostate epithelium. Conclusions.—Like hK7, ALP is down-regulated in prostate cancers, which begs the question of whether it remains an effective inhibitor of hK7 or whether it is discordant in time or space and is ineffective as an inhibitor of hK7. The function of KLK7 and ALP in prostate cancer should be further studied.

Ischemia-induced receptor for activated C kinase (RACK1) expression in rat kidneys

1997 ◽  
Vol 272 (2) ◽  
pp. F160-F166 ◽  
Author(s):  
B. J. Padanilam ◽  
M. R. Hammerman
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
Differential Display ◽  
Renal Injury ◽  
Proximal Tubules ◽  
Chain Reaction ◽  
Tubular Lumen ◽  
S3 Segment ◽  
Polymerase Chain ◽  
Ischemic Renal Injury

Differential display-polymerase chain reaction (DD-PCR) was used to identify genes that are expressed in kidney following induction of acute ischemic renal injury. The receptor for activated C kinase (RACK1) mRNA expression in kidneys obtained from rats 12 h following ischemia is enhanced twofold compared with sham-operated rats. The maximal enhancement of expression (3.3-fold) is at 7 days following reperfusion. Expression remains elevated at 14 days. RACK1 transcripts and protein are localized to the damaged and regenerating segments of proximal tubules. At 1 day following injury, RACK1 protein is present in the epithelial cells of the damaged S3 segment and in cells sloughed into the tubular lumen. By 5 days following injury, RACK1 protein expression is enhanced in the regenerating cells relining the injured tubules of the S3 segment and in papillary proliferations within regenerating tubules. Increased expression of RACK1 could enhance the activity of PKC and, in so doing, regulate the process of regeneration of the proximal tubule following ischemic renal injury.

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.

scholarly journals Clinical Value and Potential Mechanisms Exploration of GOLGA8B in Hepatocellular Carcinoma: A Comprehensive Investigation Based on Real-Time Quantitative Polymerase Chain Reaction, RNA-seq, Microarray and Immunohistochemistry

2019 ◽  
Author(s):  
Fu-chao Ma ◽  
Gao-qiang Zhai ◽  
Hua-yu Wu ◽  
Rong-quan He ◽  
Qi Sun ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Polymerase Chain Reaction ◽  
Protein Expression ◽  
Rna Sequencing ◽  
Quantitative Polymerase Chain Reaction ◽  
Sequencing Data ◽  
Hub Genes ◽  
Chain Reaction ◽  
Microarray Chip ◽  
Polymerase Chain

Abstract Introduction Recent studies found that GOLGA8B plays an essential role in different cancers. However, the role GOLGA8B plays in the carcinogenesis and development of hepatocellular carcinoma (HCC) remains unclear. This study explores the clinical significance and prospective mechanisms of GOLGA8B in HCC. Materials and methods The expression of GOLGA8B was detected in tissues of HCC and non-HCC controls. A real-time quantitative polymerase chain reaction analysis was performed to evaluate the mRNA expression of GOLGA8B. RNA-sequencing data and microarray chip data were obtained for further analysis. The role GOLGA8B plays in patients with HCC was also evaluated. An immunohistochemistry (IHC) analysis was also performed to evaluate the protein expression of GOLGA8B. The different GOLGA8B expression resources, including mRNA and protein expression, were integrated by calculating standard mean difference (SMD) and summary of the receiver operator characteristic (sROC). Genes co-expressed GOLGA8B were predicted. Enrichment analyses including Gene ontology (GO) and biological pathway were performed to investigate the essential molecular mechanisms. Hub genes were screened out by a protein-protein interactions network. MicroRNAs which target GOLGA8B at a posttranscriptional level were also predicted. Results According to different resources, GOLGA8B manifested a higher expression in tissues of HCC than in non-HCC controls and exhibited clinical values for HCC. The RT-qPCR analysis revealed an increasing trend of GOLGA8B expression in HCC. GOLGA8B expresssion was significantly increased in RNA-sequencing and 7 of 13 microarray chip. IHC analysis also revealed significantly higher expression of GOLGA8B protein. Moreover, GOLGA8B expression was correlated with pathologic tumors and stages according to RNA-sequencing data and IHC analysis. The integrated SMD and sROC of different resources was 0.893 (P=0.004) and 0.79. A total 1303 co-expressed genes were gathered to perform enrichment analyses. The most significant biological pathways of co-expressed genes were spliceosome and mitogen-activated protein kinase signalling (MAPK). Four hub genes (SF3B1, HNRNPA2B1, HNRNPA1 and SRRM2) and five miRNAs (miR-369-3p, miR-203a, miR-374b-5p, miR-139-5p and miR-144-3p) were screened out. Conclusion GOLGA8B expression was increased in HCC and may serve as a novel target for HCC diagnosis and treatment.

scholarly journals Inactivation of parkin by promoter methylation correlated with lymph node metastasis and genomic instability in nasopharyngeal carcinoma

Tumor Biology ◽  
2017 ◽  
Vol 39 (3) ◽  
pp. 101042831769502 ◽  
Author(s):  
Haifeng Ni ◽  
Zhen Zhou ◽  
Bo Jiang ◽  
Xiaoyang Yuan ◽  
Xiaolin Cao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nasopharyngeal Carcinoma ◽  
Protein Expression ◽  
Mrna Expression ◽  
Genomic Instability ◽  
Promoter Methylation ◽  
Chain Reaction ◽  
Node Metastasis ◽  
Mrna And Protein Expression ◽  
Polymerase Chain

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

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