Reverse Transcription Multiplex Ligation-dependent Probe Amplification (RT-MLPA) assay
Department of Infectious Diseases - Leiden University Medical Center, Leiden, The Netherlands
To investigate the human immune response to newly developed or existing vaccines, or during infection/disease on a population scale, the Department of Infectious Diseases at LUMC has developed a dual-color Reverse Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) assay, which can rapidly profile mRNA expression of host genes. dcRT-MLPA is a fast, reliable, robust, low cost and user friendly technique permitting mRNA expression profiling of as many as 90 loci in a single reaction.
dcRT-MLPA is based upon the ligation of two chemically synthesised half-probes when hybridised adjacently to a target cDNA sequence. Ligation is facilitated by 5’ phosphorylation of the right hand half-probe. dcRT-MLPA is designed in such a way that all ligated fragments are amplified with the use of only two fluorophor-conjugated PCR primer pairs. This PCR amplification step within dcRT-MLPA ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. The length of each amplification product is unique and increases in a stepwise fashion by only 2 nucleotides, with a total size range lying between ~80-220 nucleotides. This size range provides optimal fragment separation and a low background using capillary electrophoresis. Fluorescence-intensity of each amplification product (peak area) is quantified using GeneMapper software package and category tables containing peak areas are exported for further bioinformatics analysis. Significant differences between study groups are determined using Kruskal–Wallis H and Dunn’s multiple comparison tests. To determine biomarker signatures with the highest discriminatory power, the data sets are analysed using the Lasso regression model, which is a shrinkage and selection method for linear regression.
Using dcRT-MLPA, genes of interest can be selected on a tailor-made basis and are interchangeable. Since this assay is high throughput (96 well format) and only requires 100 ng RNA per sample, it is an exceptionally suitable technique for performing semi-large scale gene expression analyses. We have found dcRT-MLPA has a dynamic range and sensitivity very close to qRT-PCR (SA Joosten et al, Genes & Immunity 13(1):71-82, 2011). Recent work probing differences in immune gene expression activity in populations yielded new candidate biomarkers associated with tuberculosis disease and the response to chemotherapy (ibid, and JS Sutherland et al, PLoS One 6(9):e25230, 2011). Thus, dcRT-MLPA will allow analysis of host immune gene expression in relation to vaccination, infection, co-infection, disease or therapeutic interventions. dcRT-MLPA can also be used to probe differences in immune reactivity in populations in (and between) different areas, e.g. due to environmental or genetic factors which may impact on vaccine efficacy, such as co-infections with helminths.
We have selected genes to assess human adaptive and innate immune responses. The versatility of the RT-MLPA assay allows incorporation of any new gene of interest, which can be selected on a tailor-made basis). We have the following sets of genes operational:
- gene set to monitor the adaptive immune response (adaptive immune profiling):
- T cell subsets (CD3, CD4, CD8, CCR7, CD45RO, CD45RA).
- Th1 responses (CXCL10, IFNG, IL12, IL15, IL1B, IL2, TBX21, TNF).
- Th2 responses (GATA3, IL10, IL13, IL4, IL5, IL6, IL9, TGFB1).
- Th17/22 responses (IL17, IL22RA1, IL23A, RORC, genes we have recently identified by microarray).
- Treg responses (CCL4, CTLA4, FOXP3, IL2RA, IL7R, LAG3, GITR).
- T cell cytotoxicity (GNLY, GZMA, GZMB, PRF1).
- B cell associated genes (CD19, FCGR1A, BLR1).
- gene set to monitor the innate immune response (innate immune profiling):
- Key genes in MF1 (pro-inflammatory) and MF2 (anti-inflammatory / alternatively activated) macrophages: IL12A, IL12B, IL23A, CCL5, CCL22, IL10, CCL2, CXCL13, other genes we have recently identified by microarray.
- Pattern recognition receptors (TLR1-10).
- Co-receptors (MRC1-2, CD209, CLEC7A).
- Other PRR and inflammasome related genes (NLRP1-4, 6, 7, 10-13, NOD1-2, NLRC4).
MLPA training can be provided to individual sites if required
Modalities of access
Organisation short name: LUMC
Unit of access: The unit of access is the running of dcRT-MLPA assay and data delivery (see above).
Quantity of access available: A maximum of 1000 studies are offered.
Timelines: On basis of availability
Overview of access provided between January 2011 (1st call) and January 2013 (9th call):
Estimated number of users/User groups: On basis of availability
Estimated number of projects: On basis of availability
Access already provided: 0 (This service has been included to the TRANSVAC network in July 2012)
Access still available: On basis of availability
Last call submission date scheduled for: April 2013 (Call number: 10)
Input – sample requirements
Users can submit isolated RNA samples for bioprofiling to our facility.
Output – deliverables to User
dcRT-MLPA assay and fragment runs will be performed using a thermocycler and 96-capillary sequencer, respectively. Fluorescence-intensity of each amplification product (peak area) will be quantified using GeneMapper software package and category tables containing peak areas will be exported to an Excel spreadsheet (that automatically normalizes the data for the included 4 reference genes) for further bioinformatics analysis by the users.
Tom HM Ottenhoff
t [dot] h [dot] m [dot] ottenhoff [at] lumc [dot] nl
Marielle C Haks
m [dot] c [dot] haks [at] lumc [dot] nl
+31 71 5262620