Transcriptome analysis with and without reference
From the tissue to the transcriptome
Transcriptome or expression analyses can be used to identify genetic functions and physiological signal paths and thus increase the understanding of the role of the genes in growth regulation, differentiation, apoptosis, development of cancer and neurogenesis, for example. The use of microarrays is currently the most popular technique for gene expression studies but one which is limited because it only detects known RNA transcripts contained on the array.
The use of Next Generation sequencing technologies, on the other hand, also permits the analysis of complete transcripts (cDNA) with or without reference:
- Normalized, subtracted, prokaryotic
- Complete amplicons or exons
- Quantification (tag profiling; SAGE, CAGE,...)
Project example:
Identification of transcriptomes which are not detectable with commercial arrays
Project Design and Results:
1. Pool polyA+ from all four samples and generate a normalised cDNA library
2. Sequence the cDNA using the GS FLX Titanium protocol
3. Cluster the resulting sequences
4. Blast the cluster representatives against the known ESTs
5. Generate a non-normalised cDNA library from the four samples using four barcodes
6. Sequence the four tagged pooles samples on the Genome Analyzer II (36bp single end)
7. Map the Genome Analzyer II reads to the GS FLX sequence data
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