Stripping custom microRNA microarrays and the lessons learned about probe–slide interactions

Zhang, Xiaoxiao; Xu, Wayne; Tan, Jiankang; Zeng, Yan

doi:10.1016/j.ab.2008.12.008

Cited by 8 publications

(11 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ECs were grown either in normoxia or hypoxia for different periods of time (24 or 48 hours) and miRNA levels were determined by custom arrays (13). Details of normalization and statistical methods used to determine changes in expression levels have been previously published (13). For array analyses of the same RNA sample, Pearson correlation coefficients were 0.90-0.99, demonstrating good reproducibility of our microarray experiments (13).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Ghosh¹,

Subramanian²,

Adhikari³

et al. 2010

J. Clin. Invest.

Self Cite

391

288

View full text Add to dashboard Cite

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Ghosh¹,

Subramanian²,

Adhikari³

et al. 2010

J. Clin. Invest.

Self Cite

391

288

View full text Add to dashboard Cite

show abstract

“…We then investigated whether micro-RNA arising from DNM2os could be involved in feedback regulation of HIF. A custom microarray was used (10). The levels of several miRNAs were altered in hypoxic EOCCs ( Fig.…”

Section: Significancementioning

confidence: 99%

Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis

Joshi¹,

Subramanian²,

Schnettler³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominantnegative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3′-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR199a, and HIF, with implications in cancer metastasis.microRNA | iron regulation E pithelial ovarian cancer (EOC) is the leading cause of death among the gynecological malignancies (1). Ascites development and peritoneal metastasis are unique features of ovarian cancer progression. Gas analyses of ovarian cancer ascites show about 2.5% dissolved oxygen content, whereas the blood oxygen content ranges between 15% and 23% (2). Hypoxic areas are common in tumor microenvironment as increased metabolic demands of rapidly proliferating cells outpace oxygen availability. Sustained exposure to hypoxia spurs cells to reorganize cellular processes, and energy-consuming functions, such as endocytosis, are suppressed (3, 4). Hypoxia-inducible factor-1α and hypoxia-inducible factor-2α (HIF-1α/HIF-2α) are principal coordinators of these responses. HIF-1α/HIF-2α are stabilized in hypoxia and associate with hypoxia-inducible factor-1β (HIF-1β) to form heterodimeric transcription factors and induce the expression of target genes (5, 6). HIF-1-mediated expression of lysyloxidase (LOX) cross-links collagens and induces cell migration (7). Epithelial ovarian cancer cells (EOCCs) that have adapted to hypoxia by activating HIF-1 disseminate from primary ovarian tumors and exfoliate into the peritoneal cavity. HIF-1 significantly enhances gene signatures associated with tissue remodeling, the morbidity and mortality associated with EOC (8, 9).Regulation of HIF-1 is a key step in the hypoxic response with profound implications for EOC metastasis. Under normoxia, HIF-1α is hydroxylated within its oxygen-dependent degradation domain (ODDD) by prolylhydroxylases (PHDs). This reaction is an oxygen-, iron-, 2-oxoglutarate and ascorbate-dependent process. Hydroxylated HIF-1α is recognized and bound by a complex that...

show abstract

“…Printing microarrays in bulk and reusing slides and lifterslips will significantly reduce the cost. Reusing slides has the additional advantage of improving the consistency of microarray experiments 7 . We have reused slides more than six…”

Section: Discussionmentioning

confidence: 99%

“…5. Once satisfactory signals are obtained after 4.3), strip the microarray slides for reuse 7 . Rinse the slides in water, then immerse in pre-warmed, 1 mM NaOH and 0.1 x SSC in a staining dish at 62°C for 10-20 minutes.…”

mentioning

confidence: 99%

Performing Custom MicroRNA Microarray Experiments

Zhang

Zeng

2011

JoVE

Self Cite

View full text Add to dashboard Cite

microRNAs (miRNAs) are a large family of˜22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1 . Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3 . miRNAs control a broad range of biological processes 1 . In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4,5 . It is important, therefore, to understand the expression and functions of miRNAs under many different conditions. Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

show abstract

Stripping custom microRNA microarrays and the lessons learned about probe–slide interactions

Cited by 8 publications

References 30 publications

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis

Performing Custom MicroRNA Microarray Experiments

Contact Info

Product

Resources

About