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Multiplexed Detection of Cytokine Cancer Biomarkers using Fluorescence RNA In Situ Hybridization and Cellular ImagingDownload
February 03, 2016
Authors: Brad Larson, BioTek Instruments, Inc. Winooski, VT USA; Dylan Malayter and Michael Shure, Affymetrix, Inc. Santa Clara, CA USA
Cytokines play an important role in multiple aspects of cancer, including development and advancement, treatment, and prognosis. Within the tumor environment they contribute to tumorigenesis, tumor progression, and apoptosis. Expression of specific cytokines has also been implicated in enhanced tumor cell survival rates as well as metastatic activity. While many cancer related cytokines have been identified, the pro-inflammatory cytokines IL-6 and IL-8 have been linked to a wide range of cancers including lymphoma, melanoma, breast, prostate, and colorectal cancers, among others. Specifically, increased expression of IL-6 has been seen in patients with colorectal (Komoda, et al., 1998) and prostate (Culig, et al., 2012) cancers. IL-8 is also expressed in prostate cancer cells, where its presence has been linked to the metastatic potential of these cells (Aalinkeel, et al., 2004). This same role was also identified for breast cancer, where high levels of IL-8 expression increase the invasiveness estrogen-receptor negative breast cancer cells (Freund, et al., 2003). Therefore profiling cytokine expression can be an important method as a diagnostic tool and predictor of cancer prognosis.
Fluorescence based in situ hybridization (FISH) techniques have become a common method to visualize nucleic acid expression at the DNA or RNA level within cells. However the fluorescence in situ hybridization of RNA has always been limited by low sensitivity, complicated workflow and the inability to perform multiplex analysis. Here we describe a unique, non-radioactive RNA in situ hybridization solution that offers single-molecule RNA sensitivity and multiplexed analysis for one to four RNA targets. The fluorescence emanating from the amplified signal associated with each mRNA molecule can be easily captured using a novel cell imaging multi-mode reader. With up to four fluorescence imaging cubes capable of being installed in the instrument, simultaneous detection of the multiplexed assay can be accomplished. Levels of RNA expression are then determined using cellular analysis algorithms to identify the number of mRNA molecules per cell in each image. The combination provides an efficient, sensitive and repeatable method to test for the presence of important predictive cancer biomarkers.
Cytation™ 5 Cell Imaging Multi-Mode Reader