Vala Sciences

All gene expression begins with transcription. As the process of transcribing messenger RNAs from the genome is heavily regulated, the number of copies of mRNA present in a cell transcribed from a single gene can vary from none to well over a thousand. Through use of inexpensive reagents, simple assay protocols and effective software, the location and number of individual mRNA species can now be visualized and quantified in cells and tissue sections (Figure 1). This novel technology has broad application across many processes from cell differentiation to response to hormones, drugs and disease.

Affymetrix Quantigene® ViewRNA probes bind to target mRNA species with very high specificity. Probes can be generated to any known sequence and are hybridized to the target mRNAs in fixed and permealized cell or tissue samples. A fluorescent reagent is then added, which binds to the Affymetrix Quantigene® ViewRNA probe. When slides and well plates containing cultured cells are processed in this manner, and visualized with fluorescence microscopy, bright mRNA loci are apparent as spots in the image that correspond to individual copies of the target mRNA (Figure 2). To quantify gene transcription, the mRNA loci are counted for each cell.

Visualization and quantification of mRNA by Affymetrix Quantigene® ViewRNA assay and Vala’s CyteSeer® software. Left, HeLa cells visualized for nuclei (blue) and mRNA (green). Right, mRNA loci identified by CyteSeer®.

Vala’s CyteSeer® provides a reliable automated software solution to accurately quantify these mRNA spots (Figure 2). Cell nuclei are identified using images of a nuclear counterstain (e.g., DAPI or Hoescht) while mRNA spots are identified using green fluorescent labeled spots from the Quantigene® ViewRNA assay. The assay and software can be used with most digital fluorescent microscopy and high-content screening systems. All nuclear and mRNA loci are quantified on a per cell basis. CyteSeer® is specifically engineered for Quantigene® ViewRNA and is available for Windows, Mac and Linux operating systems. Results from CyteSeer® are highly tunable and are readily accessible by many spreadsheet and other data analysis tools.

Quantification of induction of IL-8 mRNA by PMA with the Quantigene® ViewRNA assay and CyteSeer® software. Left, visualization of mRNA loci (green) and nuclei (blue) for cells exposed to 1 ng/ml PMA. Middle, mRNA loci identified by CyteSeer®. Right, Dose-response relationship for induction of IL-8 mRNA by PMA. Each bar represents the mean of 67 to 100 cells.

To illustrate the performance of the Quantigene® ViewRNA assay and CyteSeer® quantification in an experimental setting, cells were exposed to different concentrations of phorbol 12-myristate 13-acetate (PMA), and analyzed for the copy number of IL-8 mRNA. For control cells (exposed to 0 PMA), essentially no IL-8 mRNA were detected (0.05/cell) by CyteSeer®. In contrast, exposure to PMA led to a dramatic increase in the presence of the loci with an EC50 of between 0.1 and 1 ng/ml PMA (Figure 3).

The potential applications of Quantigene® ViewRNA & CyteSeer® technology are numerous. The assay provides a quantitative readout of transcription, a process that is fundamental to virtually all cell and molecular biology and disease states. These techniques are broadly useful for individual academic researchers wishing to closely quantify the transcriptional activity of cells under various conditions of interest as well as by pharmaceutical companies wishing to screen large compound libraries for potential therapeutic effects.

For more information on this amazing and novel methodology, please contact the following:

Vala Sciences Inc:
Sales and information:
858 481-6862
http://valasciences.com/

Affymetrix:
Sales and information:
877 PANOMIC (1-877-726-6642)
http://Affymetrix.com/