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Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue.

ABSTRACT: BACKGROUND: Low density arrays (LDAs) have recently been introduced as a novel approach to gene expression profiling. Based on real time quantitative RT-PCR (QRT-PCR), these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to QRT-PCR. We have now evaluated LDAs as a means of determining the expression of multiple genes simultaneously in human tissues and cells. RESULTS: Comparisons between LDAs reveal low variability, with correlation coefficients close to 1. By performing 2-fold and 10-fold serial dilutions of cDNA samples in the LDAs we determined a clear linear relationship between the gene expression data points over 5 orders of magnitude. We also showed that it is possible to use LDAs to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of 1 x 10(2)-1 x 10(6) copies. Furthermore, the data generated by the LDA from a cell based pharmacological study were comparable to data generated by conventional QRT-PCR. CONCLUSION: LDAs represent a valuable new approach for sensitive and quantitative gene expression profiling.

SUBMITTER: Goulter AB

PROVIDER: S-EPMC1403755 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue.

Goulter Andrew B AB Harmer Daniel W DW Clark Kenneth L KL

BMC genomics 20060224

<h4>Background</h4>Low density arrays (LDAs) have recently been introduced as a novel approach to gene expression profiling. Based on real time quantitative RT-PCR (QRT-PCR), these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to QRT-PCR. We have now evaluated LDAs as a means of determining the expression of multiple genes simultaneously in human tissues and cells.<h4>Results< ...[more]

PMID: 16504128

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Project description:Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450?K to assess methylation profile along with 390?MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450?k. Similarly, we tested this simplified set of gene signatures in 763?MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k?=?4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making.

Dataset Information

Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue.

Publications

Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue.

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