Targeted transcript profiling by sequencing
(English)Manuscript (preprint) (Other academic)
In recent years, second generation sequencers have been employed to study various facets of the transcriptome in a comprehensive manner. However, intermediary gene sets featuring differentially expressed genes can reduce the dimensionality of experiments while providing researchers with the most significant data. Trinucleotide threading (TnT) is a multiplex amplification method previously implemented in an assay for expression profiling of moderate gene sets. Here, two additional detection systems were evaluated with a focus on lowering the input material requirements. 32 genes were simultaneously assayed with detection either by direct hybridization of TnT products or by sequencing these using the massively parallel 454 sequencer. Both approaches produced reliable transcript abundance data starting from total RNA from about 200 cells. The direct hybridization readout is beneficial for smaller-scale studies, while more ambitious efforts employing numerous individuals are, together with a sample barcoding and pooling scheme, well suited for the second generation sequencing approach. Moreover, with protocol optimizations the starting material requirements for the sequencing strategy may be further reduced. Accordingly, this study presents a targeted RNA-Seq method.
Other Industrial Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-11293OAI: oai:DiVA.org:kth-11293DiVA: diva2:272179
QC 201008192009-10-142009-10-142011-02-04Bibliographically approved