Development of an ultra-high throughput long non-coding RNA qPCR screening system

Jan Hellemans1, Pieter Mestdagh2, Steve Lefever2, Barbara D’haene1, Filip Pattyn2, Frank Speleman2, Jo Vandesompele1
1Biogazelle, Ghent, Belgium; 2Ghent University, Ghent, Belgium

Long non-coding RNAs (lncRNA) are an underexplored class of non-coding RNAs and have been shown to be implicated in health and disease. They constitute a new class of biomarkers with disease associated lncRNA signatures yet to be discovered. In addition, they open up a new approach to understand the function and organization of the genome. The lack of a high-throughput platform to detect and quantify lncRNAs has hampered their study so far. To address this, we developed a new platform for ultra-high throughput RT-qPCR analysis of long non-coding RNAs. In the pilot phase, we designed qPCR assays for +1000 lncRNAs based on public sequence databases and lincRNA chromatin signatures. All assays were designed using state-of-the-art in silico quality controls, followed by extensive empirical validation according to MIQE guidelines. Validation of this first set in relevant biological model systems demonstrated excellent sensitivity and specificity of the technology. Data processing was done using the qbasePLUS software with an improved global mean normalization procedure to better remove technical variation. In conclusion, we have successfully completed the first stage of the development process of an ultra-high throughput and low-volume RT-qPCR platform for the quantitative detection of lncRNAs. The tool offers a unique way to investigate the expression patterns of lncRNAs in health and disease.

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