Influence of RNA Labeling on High-Throughput Expression Profiling of MicroRNAs
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Kaddis, John S, Bowers, Jessica, Wai, Daniel H, Hartmann, Nicole, Baeriswyl, Lukas, Bajaj, Sheetal, Anderson, Michael J, Getts, Robert C and Triche, Timothy J (2012) Influence of RNA Labeling on High-Throughput Expression Profiling of MicroRNAs. The Journal of Molecular Diagnostics, 14 (1). pp. 12-21. ISSN 1525-1578
Abstract
Background:
Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown if changes made to labeling reagents or components used during the labeling step affect the starting RNA requirements or microarray performance.
Methodology:
Human brain/lung samples were each labeled in duplicate, at 1.0ug, 0.5ug, 0.2ug, and 0.1ug of total RNA, using two microRNA labeling kits that utilize the same labeling procedure but differ in the composition of the labeling reagent used to label the microRNA molecules in the samples. Statistical measures of reliability and validity were used to evaluate microarray data. Cross-platform confirmation was accomplished using TaqMan microRNA assays. Synthetic microRNA spike-in experiments were also performed to establish the signal dynamic range of the microarray using the ligation-modified kit.
Results:
Technical replicate correlations of signal intensity values were high using both kits, but improved with the ligation-modified assay. The drop in detection call sensitivity and gene list correlations observed using reduced amounts of standard-labeled RNA was considerably improved with the ligation-modified kit. Microarray signal dynamic range was found to be linear across 3 orders of magnitude from 4.88 to 5000 attomoles.
Significance:
Our results show that optimization of the microRNA labeling reagent can result in at least a 10-fold decrease in microarray total RNA requirements with little compromise to data quality. Clinical investigations bottlenecked by the amount of starting material may employ a ligation mix modification strategy to reduce total RNA requirements.
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| Item Type: | Article |
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| Additional Information: | Spike-in validation concept tested here is considered to be extended into other assays incl. NGS |
| Keywords: | microRNA array, Genisphere® FlashTag™ biotin-HSR (biotin-HSR) assay, TaqMan miRNA assay |
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| Date Deposited: | 13 Oct 2015 13:15 |
| Last Modified: | 13 Oct 2015 13:15 |
| URI: | https://oak.novartis.com/id/eprint/5078 |