Focused human gene expression profiling using dual-color reverse transcriptase multiplex ligation-dependent probe amplification.

To investigate the human immune response to newly developed or existing vaccines, or during infection/disease on a population scale, we have recently developed a dual-color Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) assay, which can rapidly profile mRNA expression of multiple host genes. dcRT-MLPA has a dynamic range and sensitivity comparable to real-time QPCR and RNA-Sequencing. Since this assay is high-throughput, it is an exceptionally suitable technique for monitoring host biomarkers in semi-large scale human cohorts, such as cross sectional studies with multiple groups, or longitudinal studies with multiple time points. Multicomponent host biomarker signatures with excellent predictive values can easily be identified using lasso regression analysis, while exploring additional data adjustment methods like RUV-2 may further optimize the identification of informative host biomarker signatures. dcRT-MLPA also allows comparisons of gene expression patterns across different human populations to explore the impact of geographical diversity on for example vaccine induced responses. The use of dcRT-MLPA is not limited to peripheral blood but can be adapted to analyze host biomarkers derived from any tissue or body fluids, further demonstrating the versatility of the dcRT-MLPA platform. Several examples will be given and discussed.