| 1. |
- Grundberg, Ida, et al.
(författare)
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In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics
- 2013
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 4:12, s. 2407-2418
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Tidskriftsartikel (refereegranskat)abstract
- Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
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| 2. |
- Weibrecht, Irene, et al.
(författare)
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In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay
- 2013
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 8:2, s. 355-372
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Tidskriftsartikel (refereegranskat)abstract
- Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.
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