Targeted transcript profiling by sequencing
2012 (English)In: Scientific Reports, ISSN 2045-2322, Vol. 2, 821- p.Article in journal (Refereed) Published
In this work we present a targeted gene expression strategy employing trinucleotide threading (TnT) amplification and massive parallel sequencing. We have previously shown that TnT combined with array readout accurately monitors expression levels. However, with this detection strategy spurious products go undetected. Accordingly, we adapted the TnT protocol to massive parallel sequencing to acquire an unbiased view of the entire TnT-generated product population. In this manner we investigated the identity of undesired products, their extent at different oligonucleotide: RNA ratios and their effect on the expression levels. We demonstrate that TnT gene expression profiling with massive sequencing readout renders reliable expression data from as low as 3.5 ng of total RNA. Moreover, using 350 ng of total RNA results in only 0.7% to 1.1% undesired products. When lowering the amount of input material, the undesired product fraction increases but this does not influence the expression profiles.
Place, publisher, year, edition, pages
2012. Vol. 2, 821- p.
Gene-Expression, Drug Discovery, Technologies
IdentifiersURN: urn:nbn:se:kth:diva-107616DOI: 10.1038/srep00821ISI: 000310879000001ScopusID: 2-s2.0-84869151594OAI: oai:DiVA.org:kth-107616DiVA: diva2:577741
FunderKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 201212172012-12-172012-12-142012-12-17Bibliographically approved