Abstract
BackgroundIn clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated. This approach reduces costs and complexity of data analysis and allows multiplexing of samples. Polymerase-chain-reaction (PCR) based TaqMan assays have high sensitivity but suffer from a limited dynamic range and sample throughput. Hence, there is a gap for a technology able to measure expression of large gene sets in multiple samples.ResultsWe have adapted a commercially available mRNA quantification assay (AmpliSeq-RNA) that measures mRNA abundance based on the frequency of PCR amplicons determined by high-throughput semiconductor sequencing. This approach allows for parallel, accurate quantification of about 1000 transcripts in multiple samples covering a dynamic range of five orders of magnitude. Using samples derived from a well-characterized stem cell differentiation model, we obtained a good correlation (r = 0.78) of transcript levels measured by AmpliSeq-RNA and DNA-microarrays. A significant portion of low abundant transcripts escapes detection by microarrays due to limited sensitivity. Standard quantitative RNA sequencing of the same samples confirms expression of low abundant genes with an overall correlation coefficient of r = 0.87. Based on digital AmpliSeq-RNA imaging we show switches of signaling cascades at four time points during differentiation of stem cells into cardiomyocytes.ConclusionsThe AmpliSeq-RNA technology adapted to high-throughput semiconductor sequencing allows robust transcript quantification based on amplicon frequency. Multiplexing of at least 900 parallel PCR reactions is feasible because sequencing-based quantification eliminates artefacts coming from off-target amplification. Using this approach, RNA quantification and detection of genetic variations can be performed in the same experiment.
Highlights
In clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated
Workflow of AmpliSeq-based digital transcript imaging (AmpliSeq-RNA) Compared with DNA genotyping, the AmpliSeq-RNA quantification workflow (Figure 1) requires polyA-primed conversion of mRNA to cDNA followed by target specific PCR amplification using a multiplex primer pool
For experimental validation of AmpliSeq-RNA we have compiled a custom panel of 917 transcripts representing about 150 biological signaling cascades
Summary
In clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated. This approach reduces costs and complexity of data analysis and allows multiplexing of samples. Robust and validated PCR-based assays interrogating disease specific single-nucleotide-polymorphisms (SNP) panels are routinely used in exploratory and clinical research using formalin fixed-paraffin embedded (FFPE) or biopsy derived DNA samples. Expressed transcripts found in 3D-cultures, human primary cells or clinical samples are useful resources for generation of customized assays interrogating expression of disease relevant genes only. The microfluidics based quantitative PCR (qPCR) platform (“Fluidigm”) allows multiparallel expression analysis of 96 custom transcripts in 96 samples at high sensitivity with low RNA input [1]. All technologies briefly outlined above suffer either from low sample throughput or else from a rather limited number of transcripts for multiparallel analysis
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