Monday, December 23, 2013

Image Based Detection of RNA in Live Cells

The determination of intracellular RNA levels is a critical component in elucidating the cellular responses of living cells to external stimuli. Many of the techniques traditionally used for RNA quantitation involve transfection, laborious sample preparation and RNA amplification, which can preclude large sample numbers. However, disease directed research, which often involves the screening of compound libraries, relies on the ability to rapidly make assay determinations on large numbers of samples. At the same time phenotypic information is also desired to assess the true cellular response. Towards that end, having multiple fluorescent probes capable of simultaneously detecting different cellular RNAs in live cells are of particular importance.

SmartFlare™ Structure.

Figure 1.  SmartFlare Structure.

SmartFlare™ RNA detection probes consist of a gold nanoparticle conjugated to multiple copies of a double-stranded oligonucleotide. One of the two strands (capture strand) is covalently linked to the gold nanoparticle, while the other strand (reporter strand), which is complimentary to the capture strand has an attached fluorophore that is quenched by proximity to the gold nanoparticle core. When the SmartFlare nanoparticle comes in contact with its analyte RNA-target, the RNA binds to its complimentary capture strand and displaces the reporter strand. The free reporter strand, whose fluorophore is no longer quenched, can be detected via fluorescence.

These probes can identify cell line expression of specific gene. For example when the breast cancer cell lines MCF-7 and SK-BR-3 are probed with the ERBB2 probe, only the SK-BR-3 cell line expresses large amounts of the RNA. Live MCF-7, SK-BR-3 or a 50/50 mixture of the two cell lines treated with CY3-GAPDH and CY5-ERBB2 probes were imaged using a Cytation 3 Imaging plate reader. Subpopulation analysis of DAPI stained objects in Gen5 software was used to indentify of CY3 and CY5 positive cells. As demonstrated in Figure 2, all of the DAPI strained objects (cell nuclei) were also positive for the housekeeping gene glyceraldehydes 3-phosphate dehydrogenase (GAPDH), while the fraction of positive cells for ERB-B2 is dependent on the fraction of SK-BR-3 cells present. Furthermore, these data also demonstrate the ability of the technology, (chemistry and imager) to multiplex two separate RNA species simultaneously.

Subpopulation analysis of images of MCF-7 and SK-BR-3 cells treated with SmartFlare probes.

Figure 2. Subpopulation analysis of images of MCF-7 and SK-BR-3 cells treated with SmartFlare probes. 

These data are further corroborated by live cell antibody staining. When live MCF-7 and SK-BR-3 cells are stained with Hoechst 33342 dye and an Alexa Fluor® 488 labeled anti-EGFR antibody and imaged with a Cytation 3 Imaging Microplate reader only SK-BR-3 cells exhibit green fluorescence (Figure 3). Most notably the fluorescence is limited to the cell surface indicating the live cells have intact viable cell membranes.

Anti-EGFR antibody staining of Live MCF-7 and SK-BR-3 cells.

Figure 3.  Anti-EGFR antibody staining of Live MCF-7 and SK-BR-3 cells.  

Do you have a need to rapidly detect RNA species in live cells or want to image live or fixed and stained cells? The combination of SmartFlare probes and the Cytation 3 Imaging microplate reader may be the answer.

By: BioTek Instruments, Paul Held, PhD., Laboratory Manager

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