Principle of Whole Genome DASL
The method of WG-DASL is an adaptation of the proven GoldenGate genotyping technology. It's principle steps involve
- A combined reverse transcription of RNA using random and oligo dT primers
- Isolation of the newly synthesized cDNA molecules
- Annealing of more than 24.000 primer pairs (20-25 transcript-specific bases per oligonucleotide, incl. universal tails) in parallel
- Primer extension and ligation to generate PCR templates
- Isolation of PCR templates
- Universal PCR facilitating amplification and fluorescent labeling
- Isolation of labeled molecules
- Hybridization to human Sentrix-8 BeadArray
Several features make WG-DASL a robust technology that can be applied to genome-wide expression profiling of partially degraded RNA in larger scale.
- Amount of input material needed is limited (>300 ng) and can be extracted from FFPE tissue
- DASL method accepts RNA, even when fragmented to <500 bases
- Robustness of technology due to large number of technical replicates on each array (av. 30 ientical spots per array)
- PCR amplified templates are short (<50-100 bp), avoiding to much bias
- Target array show a minimum of lot-to-lot variation
- Initial tests indicate high concordance with qPCR
- Many samples can be run in parallel
last update:
06/06/2011
back to top
