Post-analysis follow-up and validation of microarray experiments

Chuaqui, Rodrigo F.; Bonner, Robert F.; Best, Carolyn J.M.; Gillespie, John W.; Flaig, Michael J.; Hewitt, Stephen M.; Phillips, John L.; Krizman, David B.; Tangrea, Michael A.; Ahram, Mamoun; Linehan, W. Marston; Knežević, Vladimir; Emmert‐Buck, Michael R.

doi:10.1038/ng1034

Cited by 382 publications

(270 citation statements)

References 57 publications

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“…Moreover, mRNA expression sometimes does not reflect steady-state protein levels correctly. (47) The use of CD200 and CD24a now provides adequate cell signatures of the different maturation zones for growth plate sorting, as shown by two independent separation methods. The purity of the isolated cell populations is reflected by the change in expression levels of characteristic marker genes, as demonstrated by qPCR analysis for selected genes (Fig.…”

Section: Discussionmentioning

confidence: 99%

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Belluoccio¹,

Etich²,

Rosenbaum³

et al. 2010

Journal of Bone and Mineral Research

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Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate. ß

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Section: Discussionmentioning

confidence: 99%

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Belluoccio¹,

Etich²,

Rosenbaum³

et al. 2010

Journal of Bone and Mineral Research

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“…Our finding of a moderately positive correlation is somewhat surprising, given the greater dynamic range of single-gene assays. This may be due to false discovery associated with microarray analysis or low fold-changes of the selected genes, as some microarray validation studies have shown that consistency was not achievable for genes showing a ofour-fold difference in expression (Rajeevan et al, 2001;Chuaqui et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma

Dhanda

Triantafyllou

Lilogou

et al. 2015

British Journal of Oral and Maxillofacial Surgery

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“…PCR or Northern blot), this is only the first level of validation [21;22]. Far more interesting and complex, however, is the concept of biological validation [21]. Examination of the gene list showed several genes have been associated clinically with bladder cancer.…”

Section: Discussionmentioning

confidence: 99%

Template-driven gene selection procedure

Knowlton

Dozmorov

Kyker

et al. 2006

IEE Proc. Syst. Biol.

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The hierarchical clustering and statistical techniques usually used to analyze microarray data do not inherently represent the underlying biology. Herein we present a hybrid approach involving characteristics of both supervised and unsupervised learning. This approach is based on template matching in which the interaction of the variables of inherent malignancy and the ability to express the malignant phenotype are modelled. Immortalized normal urothelial cells and bladder cancer cells of different malignancy were grown in conventional two-dimensional tissue culture and in three dimensions on extracellular matrices that were either permissive or restrictive for expression of the malignant phenotype. The transcriptome represents the effects of two variables--inherent malignancy and the modulatory effect of extracellular matrix. By assigning values to each of the biological variables of inherent malignancy and the ability to express the malignant phenotype, a template was constructed that encapsulated the interaction between them. Gene expression correlating both positively and negatively with the template were observed, but when iterative correlations were carried out, the different models for the template converged to the same actual template. A subset of 21 genes was identified that correlated with two a priori models or an optimized model above the 95% confidence limits identified in a bootstrap resampling with 5,000 permutations of the data set. The correlation coefficients of expression of several genes were > 0.8. Analysis of upstream transcriptional regulatory elements (TREs) confirmed these genes were not a randomly selected set of genes. Several TREs were identified as significantly over-expressed in the sample of 20 genes for which TREs were identified, and the high correlations of several genes were consistent with transcriptional co-regulation. We suggest the template method can be used to identify a unique set of genes for further investigation. KeywordsBladder cancer; phenotype; transcriptomics; extracellular matrix; malignancy * This work was supported in part by NIH grants CA75322 (REH), DK 069808 (REH) and P20 RR1557, P20 RR17703, and P20 RR16478 (MBC) and by a grant from Cook Biotech (REH).

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Post-analysis follow-up and validation of microarray experiments

Cited by 382 publications

References 57 publications

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma

Template-driven gene selection procedure

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