Profiling of differentially expressed genes in wound margin biopsies of horses using suppression subtractive hybridization

2005 ◽  
Vol 22 (2) ◽  
pp. 157-170 ◽  
Author(s):  
Josiane Lefebvre-Lavoie ◽  
Jacques G. Lussier ◽  
Christine L. Theoret
Keyword(s):  
Gene Expression ◽  
Suppression Subtractive Hybridization ◽  
Wound Repair ◽  
Subtractive Hybridization ◽  
Laminin Receptor ◽  
Differentially Expressed ◽  
Mrna Levels ◽  
Matrix Metalloproteinase 1 ◽  
Dermatan Sulfate Proteoglycan ◽  
Wound Margin

Disturbed gene expression may disrupt the normal process of repair and lead to pathological situations resulting in excessive scarring. To prevent and treat impaired healing, it is necessary to first define baseline gene expression during normal repair. The objective of this study was to compare gene expression in normal intact skin (IS) and wound margin (WM) biopsies using suppression subtractive hybridization (SSH) to identify genes differentially expressed during wound repair in horses. Tissue samples included both normal IS and biopsies from 7-day-old wounds. IS cDNAs were subtracted from WM cDNAs to establish a subtracted (WM-IS) cDNA library; 226 nonredundant cDNAs were identified. Detection of genes previously shown to be expressed 7 days after trauma, including the pro-α2-chain of type 1 pro-collagen (COL1A2), annexin A2, the pro-α3-chain of type 6 pro-collagen, β-actin, fibroblast growth factor 7, laminin receptor 1, matrix metalloproteinase 1 (MMP1), secreted protein acidic cystein rich, and tissue inhibitor of metalloproteinase 2, supported the validity of the experimental design. A RT-PCR assay confirmed an increase or induction of the cDNAs of specific genes (COL1A2, MMP1, dermatan sulfate proteoglycan 2, cluster differentiation 68, cluster differentiation 163, and disintegrin and metalloproteinase domain 9) within wound biopsies. Among these, COL1A2 and MMP1 had previously been documented in horses; 68.8% of the cDNAs had not previously been attributed a role during wound repair, of which spermidine/spermine- N-acetyltransferase, serin proteinase inhibitor B10, and sorting nexin 9 were highly expressed and whose known functions in other processes made them potential candidates in regulating the proliferative response to wounding. In conclusion, we identified novel genes that are differentially expressed in equine wound biopsies and that may modulate repair. Future experiments must correlate changes in mRNA levels for precise molecules with spatiotemporal protein expression within tissues.

Subtractive hybridization for differential gene expression in mechanically unloaded rat heart

2006 ◽  
Vol 291 (6) ◽  
pp. H2714-H2722 ◽  
Author(s):  
Heiko Bugger ◽  
Stefanie Leippert ◽  
Daniel Blum ◽  
Peter Kahle ◽  
Bernhard Barleon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Suppression Subtractive Hybridization ◽  
Respiratory Chain ◽  
Rat Heart ◽  
Complex I ◽  
Subtractive Hybridization ◽  
Differentially Expressed ◽  
Isolated Mitochondria ◽  
Mechanical Unloading

The objective of this study was to identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. In male Wistar-Kyoto rats, mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by Northern blot analysis, real-time RT-PCR, and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Transplantation caused atrophy (heart-to-body weight ratio: 1.6 ± 0.1 vs. 2.4 ± 0.1, P < 0.001). We selected 1,880 clones from the subtractive hybridization procedure (940 forward and 940 reverse runs assessing up- or downregulation). The first screen verified 465 forward and 140 reverse clones, and the second screen verified 67 forward and 30 reverse clones. On sequencing of 24 forward and 23 reverse clones, 9 forward and 14 reverse homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (−49%, P < 0.05) and complex II (−61%, P < 0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level: −42% for complex I ( P < 0.01), −57% for complex II ( P < 0.05), and −65% for complex IV ( P < 0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, with glutamate and succinate as substrates: 85 ± 27 vs. 224 ± 32 natoms O·min−1·mg−1with glutamate ( P < 0.01) and 59 ± 18 vs. 154 ± 30 natoms O·min−1·mg−1with succinate ( P < 0.05). Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.

scholarly journals Profiling of BABA-induced differentially expressed genes of Zea mays using suppression subtractive hybridization

RSC Advances ◽  
2017 ◽  
Vol 7 (69) ◽  
pp. 43849-43865 ◽  
Author(s):  
Arun K. Shaw ◽  
Pardeep K. Bhardwaj ◽  
Supriya Ghosh ◽  
Ikbal Azahar ◽  
Sinchan Adhikari ◽  
...  
Keyword(s):  
Zea Mays ◽  
Differentially Expressed Genes ◽  
Suppression Subtractive Hybridization ◽  
Defense Mechanisms ◽  
Subtractive Hybridization ◽  
Differentially Expressed ◽  
Maize Leaves

This study aims to identify differentially expressed transcripts in BABA-primed maize leaves using suppression subtractive hybridization (SSH) strategy. Findings shed new light on the BABA potentiated defense mechanisms in plants.

scholarly journals Microarray analysis reveals novel gene expression changes associated with erectile dysfunction in diabetic rats

2005 ◽  
Vol 23 (2) ◽  
pp. 192-205 ◽  
Author(s):  
Chris J. Sullivan ◽  
Thomas H. Teal ◽  
Ian P. Luttrell ◽  
Khoa B. Tran ◽  
Mette A. Peters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Erectile Dysfunction ◽  
Splice Variants ◽  
Full Range ◽  
Diabetic Rats ◽  
Differentially Expressed ◽  
Diabetic Rat ◽  
Literature Mining ◽  
Mrna Levels

To investigate the full range of molecular changes associated with erectile dysfunction (ED) in Type 1 diabetes, we examined alterations in penile gene expression in streptozotocin-induced diabetic rats and littermate controls. With the use of Affymetrix GeneChip arrays and statistical filtering, 529 genes/transcripts were considered to be differentially expressed in the diabetic rat cavernosum compared with control. Gene Ontology (GO) classification indicated that there was a decrease in numerous extracellular matrix genes (e.g., collagen and elastin related) and an increase in oxidative stress-associated genes in the diabetic rat cavernosum. In addition, PubMatrix literature mining identified differentially expressed genes previously shown to mediate vascular dysfunction [e.g., ceruloplasmin ( Cp), lipoprotein lipase, and Cd36] as well as genes involved in the modulation of the smooth muscle phenotype (e.g., Kruppel-like factor 5 and chemokine C-X3-C motif ligand 1). Real-time PCR was used to confirm changes in expression for 23 relevant genes. Further validation of Cp expression in the diabetic rat cavernosum demonstrated increased mRNA levels of the secreted and anchored splice variants of Cp. CP protein levels showed a 1.9-fold increase in tissues from diabetic rats versus controls. Immunohistochemistry demonstrated localization of CP protein in cavernosal sinusoids of control and diabetic animals, including endothelial and smooth muscle layers. Overall, this study broadens the scope of candidate genes and pathways that may be relevant to the pathophysiology of diabetes-induced ED as well as highlights the potential complexity of this disorder.

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