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An antibody-based microarray assay for small RNA detection.

ABSTRACT: Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

SUBMITTER: Hu Z

PROVIDER: S-EPMC1435976 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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An antibody-based microarray assay for small RNA detection.

Hu Zonglin Z Zhang Aixia A Storz Gisela G Gottesman Susan S Leppla Stephen H SH

Nucleic acids research 20060413 7

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG a ...[more]

PMID: 16614443

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Project description:The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.

Dataset Information

An antibody-based microarray assay for small RNA detection.

Publications

An antibody-based microarray assay for small RNA detection.

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