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

Abstract

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.

BLR1 and FCGR1A transcripts in peripheral blood associate with the extent of intrathoracic tuberculosis in children and predict treatment outcome

“…The genes previously identified, have shown differential expression between TB patients, and healthy subjects with or without latent Mtb infection (LTBI), either in studies based on unbiased approaches or biased approaches where the markers were selected for their known/suggested role in TB pathogenesis (Supplementary Table 2). Moreover, the low volume of sample material required for the dcRT-MLPA was also an important consideration important given the limited amount of sample drawn per child per time-point940. As we did not apply any multiplicity adjustment of p-values there is an increased risk of reporting false positive findings, but this approach was deliberately chosen to reduce the risk that we might overlook any potential associations.…”

“…The dcRT-MLPA method can rapidly profile multiple host genes with a dynamic range and sensitivity comparable to real-time qPCR and RNA sequencing at a cost of 5–7 euros per sample40. The genes included in the dcRT-MLPA were pre-selected based on joint efforts in TB biomarker discovery by the partners in Bill and Melinda Gates foundation GC6 (Supplementary Table 2)9.…”

Big Data in Vaccinology: Introduction and section summaries

“…In a second chapter, Haks et al [20] describe a versatile transcriptomic platform to measure the expression of hundreds of human innate, adaptive and inflammatory immune genes. The platform [dual-color reverse transcriptase multiplex ligation-dependent probe amplification (dcRTMLPA)] can rapidly profile the expression of multiple (hundreds) host genes, has a dynamic range and sensitivity comparable to real-time QPCR and RNA-sequencing, is high throughput, and thus well suited 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, irrespective of the type of infection.…”

Novel transcriptional signatures for sputum-independent diagnostics of tuberculosis in children

“…In the present study, we have incorporated type I IFN-inducible genes and a broader panel of genes covering general inflammation, myeloid cell activation and humoral immunity, in a user-friendly and inexpensive technique; the dual-color-Reverse-Transcriptase-Multiplex-Ligation-dependent-Probe-Amplification (dc-RT MLPA). This technique has excellent abilities for profiling host biomarkers in larger sample sets, with a dynamic range and accuracy comparable to qPCR, requiring small amounts of RNA per sample 26 . Based on the knowledge gained from multiple genome-wide analyses, we aimed to find more defined transcriptional signatures in unstimulated WB of Indian children, with the ability to separate TB cases from young children symptomatic for other reasons, resembling a real-life diagnostic setting in India, the country carrying the greatest share of the global TB burden 1 .…”