BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay

Chemical genomics involves generating large collections of small molecules and using them to modulate cellular states. Despite recent progress in the systematic synthesis of structurally diverse compounds, their use in screens of cellular circuitry is still an ad hoc process 1-4 . Here, we outline a general, efficient approach called gene expression-based highthroughput screening (GE-HTS) in which a gene expression signature is used as a surrogate for cellular states, and we describe its application in a particular setting: the identification of compounds that induce the differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8 that reliably induced the differentiation signature and, furthermore, yielded functional evidence of bona fide differentiation. The results indicate that GE-HTS may be a powerful, general approach for chemical screening.To prove the feasibility of GE-HTS, we applied the method to the identification of compounds capable of inducing terminal differentiation of acute myelogenous leukemia (AML) cells. The plausibility of differentiation induction in leukemia is suggested by a rare subtype of AML known as acute promyelocytic leukemia (APL) in which treatment with all-trans retinoic acid (ATRA) results in clinical remissions through modulation of a mutated retinoic acid receptor alpha 5,6 . Unfortunately, ATRA has no clinical efficacy in other subtypes of AML 7 . But the fact that all forms of AML show a block of differentiation suggests that differentiation therapy might be possible, provided that the right compounds could be identified. The mechanism underlying such blocked differentiation is unknown in most cases; thus, it is not possible to carry out small-molecule screens against a validated target protein. Instead, a cell-based screening approach is needed. The usual approach to assaying the myeloid differentiation phenotype, however, involves a combination of visual inspection of nuclear morphology and biochemical tests, such as nitroblue tetrazolium (NBT) reduction 8 , neither of which readily lend themselves to a highthroughput screening platform. We therefore sought to use GE-HTS to identify compounds capable of inducing the differentiation program in AML without first knowing the crucial targets of this process.The first step in the GE-HTS procedure is defining the gene expression signatures of the biological states of interest. To accomplish this, we carried out oligonucleotide microarray-based gene expression profiling of pretreatment bone marrow samples derived from individuals affected with AML, as well as of fully differentiated peripheral blood neutrophils and monocytes derived from unaffected donors (gene expression data are available in Supplementary Table 1a,b online). We thereby identified genes correlated with the neutrophil-versus-AML or monocyte-versus-AML distinctions (Fig. 1). We selected from these differentiation-correlated genes a handful of marker genes that represented the diversity of myeloid differentiation yet could be easil...

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“…Other high-throughput detection methods, such as bead-based detection or 'arrays of arrays' are, in principle, feasible 9,10 .…”

Section: E T T E R Smentioning

confidence: 99%

Gene expression–based high-throughput screening(GE-HTS) and application to leukemia differentiation

et al. 2004

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“…Oligonucleotide capture probes were coupled to their respective microspheres (Table 1) following the protocol set forth by Yang et al (2001). Luminex xMAP™ bead stocks were sonicated using a Branson Ultrasonic Cleaner (Branson) at 40 kHz for 1 min to ensure an even distribution of beads in solution prior to being aliquoted.…”

Section: Methodsmentioning

confidence: 99%

Application of bead array technology to community dynamics of marine phytoplankton

Ellison

Burton²

2005

Mar. Ecol. Prog. Ser.

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Studies of the dynamics of marine microplanktonic systems have been hampered by a lack of high-throughput technologies for the simultaneous identification and quantification of the many taxa comprising such communities. To address this problem, we describe a DNA-hybridizationbased method of analysis employing bead array technology. Species-and/or genus-specific probes for 8 phytoplankton taxa were designed and applied to the microplankton community sampled from coastal waters off La Jolla, California, over an exploratory time series 10 d in length. Bulk DNA extractions from 1 l of seawater were used without PCR amplification. Taxa analyzed comprised 4 dinoflagellates, 3 diatoms, and 1 coccolithophorid. The results demonstrated that specifically targeted oligonucleotide probes can be used in this fashion with a high degree of binding-specificity, dependent on hybridization temperature, and that standard curves relative to target-cell concentration can be constructed. The use of 2 different probes for each taxon can provide added confidence that probes are taxon-specific. Further, single-species assays and multiplexed assays were generally in good agreement, as were assays of replicate seawater samples. Once sets of probes are developed for particular groups of taxa, the bead array system appears to provide a technological platform with great promise for high-throughput analyses of microplanktonic communities.

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“…Total RNA harvested from each subset was analyzed in parallel by the mirMASA assay, a liquidphase multiplex analytical approach utilizing hybridization probes for 114 miRNAs. 17,21,23 Pre-validation studies using synthetic miRNAs showed that s.d.s for replica determinations were 2-9% of the mean value. Readouts were normalized to the hybridization reaction of 5S RNA, the expression of which in tumor and normal tissues of individual patients did not differ significantly from one another (P ¼ 0.38; Student's two-sided paired t-test) in our study population.…”

Section: Human Prostate Cancer Signature By Array Analysismentioning

confidence: 99%

“…miRNA binding was quantified with Fluorokine MultiAnalyte Profiling (xMAP) flow cytometry of the Luminex platform. 21 Each set of color-coded microsphere (bead) is covalently coupled with a capture oligonucleotide and a biotin-labeled detection oligonucleotide. The 12-nt capture oligonucleotide recognizes the 3 0 end portion of the miRNA, whereas a short (8-10 nt), biotin-labeled detection oligonucleotide hybridizes to the 5 0 end of the same miRNA.…”

Section: Rna Extractionmentioning

confidence: 99%

MicroRNA profile analysis of human prostate cancers

et al. 2008

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We examined the microRNA (miRNA) expression profile of 40 prostatectomy specimens from stage T2a/b, early relapse and nonrelapse cancer patients, to better understand the relationship between miRNA dysregulation and prostate oncogenesis. Paired analysis was carried out with microdissected, malignant and non-involved areas of each specimen, using high-throughput liquidphase hybridization (mirMASA) reactions and 114 miRNA probes. Five miRNAs (miR-23b, -100, -145, -221 and -222) were significantly downregulated in malignant tissues, according to significance analysis of microarrays and paired t-test with Bonferroni correction. Lowered expression of miR-23b, -145, -221 and -222 in malignant tissues was validated by quantitative reverse transcription (qRT)-PCR analyses. Ectopic expression of these miRNAs significantly reduced LNCaP cancer cell growth, suggesting growth modulatory roles for these miRNAs. Patient subset analysis showed that those with post-surgery elevation of prostatespecific antigen (chemical relapse) displayed a distinct expression profile of 16 miRNAs, as compared with patients with nonrelapse disease. A trend of increased expression (440%) of miR-135b and miR-194 was observed by qRT-PCR confirmatory analysis of 11 patients from each clinical subset. These findings indicate that an altered miRNA expression signature accompanied the prostate oncogenic process. Additional, aberrant miRNA expression features may reflect a tendency for early disease relapse. Growth inhibition through the reconstitution of miRNAs is potentially applicable for experimental therapy of prostate cancer, pending molecular validation of targeted genes.

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BADGE, BeadsArray for the Detection of Gene Expression, a High-Throughput Diagnostic Bioassay

Cited by 102 publications

References 28 publications

Gene expression–based high-throughput screening(GE-HTS) and application to leukemia differentiation

Gene expression–based high-throughput screening(GE-HTS) and application to leukemia differentiation

Application of bead array technology to community dynamics of marine phytoplankton

MicroRNA profile analysis of human prostate cancers

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