Vala Sciences

After the imaging settings have been analyzed, the work shifts to analyzing the numerical data. CyteSeer ViewRNA uses a series of numerical data files to store the cell-by-cell and well-by-cell numerical data.

CyteSeer ViewRNA creates data files in the *.csv (comma separated value) format that can be loaded easily into Excel. A file that represents a summary for the experimental data set is created and is placed in the first level within the Destination folder (e.g., PMAvsIL8_DataTable.csv). Additionally, two data files are created within each well subdirectory. The wellname_DataTable.csv file contains a cell by cell data readout for every cell analyzed for the well (or area). The Well_name DataTable_Stats.csv file (e.g. C15_DataTable_Stats.csv) contains summary statistics for the well.

Open Excel on your computer and use the open command within Excel to open the C15_DataTable.csv file. It will be necessary to select “All Files” in the “Files of type” field within the Open menu of Excel to view and select csv files. Excel will automatically open a “workbook” – style interface and the spreadsheet cells will range from Excel addresses A1 to AA178 for C15_DataTable.csv. Note that a description of the file is automatically generated and displayed in Excel addresses A1 to B2 (e.g., Data Table: C15 Data Table. Description: Data Table for cells in well C15), and the Legend portion of the file extends from A5 to C3. A7 to A33 indicate the data type of each parameter (integer, double precision, or Boolean). B7 to B33 contain short descriptions, which are also the column headers for the data displayed in the Data Table portion of spreadsheet (A36 to AA178 for C15_DataTable.csv). C7 to C33 contain brief descriptions of each data parameter.

The “id” label (Excel address B7) is the header for column A in the Data Table; this is an integer number that is uniquely assigned to each cell in the image corresponding to well C15. Area Nm is the size of the nucleus in units of pixel area. This data was obtained by CyteSeer ViewRNA from the nuclear image. Area Rm (Area of the RNA mask) represents the total number of pixels identified as corresponding to RNA dots within the RNA image for each cell (Figure 19), and is an index of mRNA expression, and will be of considerable interest to the majority of users. Data parameters XLeft Nm, YTop Nm, Width Nm, and Height Nm refer to the x,y location of each nucleus within the nuclear image, and the width and height dimensions, which will assist users in identifying the location of each cell within the field of view. “IsBoundaryNm” can be either True or False; cells near the boundary of the image (IsBoundaryNm = True) might extend beyond the field of view, and, hence the analysis for RNA expression by CyteSeer ViewRNA may be incomplete. The IsBoundaryNm parameter can be used to sort the cells within Excel, and exclude boundary cells from further analysis, if desired. XCentroid Nm and YCentroid Nm are the x and y coordinates within the image for the center of each nucleus.
    
RNA spot count, Mean RNA Spot Area, and RMS RNA Spot Diameter are important data parameters relating to RNA expression. RNA spot count is the number of mRNA loci for each cell. Mean RNA Spot Area is the average size of the RNA spots for a particular cell (in units of pixel area). RMS RNA Spot Diameter is an estimate of the mean diameter of the RNA spots in the cell (RMS stands for a Root Mean Square, and refers to the method used to estimate spot diameter). Area X Nm is the area of the nucleus that is NOT also part of the RNA mask; similarly, Area X Cm is the area of the cytoplasmic mask that is NOT also part of the RNA mask. Area X Nm and Area X Cm define the size of the “background” areas within the nucleus and cytoplasm. Advanced users may find these data parameters useful, especially with comparisons to the Area Rm; for example, it might be of interest to calculate: Area Rm /(Area X Nm + Area X Cm +Area Rm), which is the ratio of the area of the RNA spots to the entire area of the cell.

Total integrated intensity of the RNA image for the RNA mask is the sum of intensities of the pixels that have been assigned to the RNA mask for each cell (TII Ri Rm - line 22 of the Legend, and column P of the Data Table), and it is another very important parameter related to mRNA expression Similarly, the average and median pixel intensities of the RNA image for the RNA mask for the cell are the API Ri Rm, and MPI Ri Rm, respectively. The Standard Deviation of Pixel Intensities for the RNA image RNA mask (SPI Ri Rm) is also reported. This parameter may be of special interest to researchers performing screens of chemical or RNAi libraries involving thousands of samples, as standard deviations of intensity can sometimes be less variable than the means or total integrated intensity measurements.

Finally, a series of data parameters are reported that correspond to the background pixel intensities. These include the total integrated, average, and median pixel intensities for the RNA image for pixels within the nuclear mask that are NOT RNA spots (TII Ri X Nm, API Ri X Nm, MPI Ri X Nm, where “X” means NOT RNA spots). The same series of values are also reported for the regions of the cytoplasm that are NOT RNA spots (TII Ri X Cm, API Ri X Cm, MPI Ri X Cm). These data parameters can be used, in combination with the data parameters for the RNA spots to quantify how bright the spots are with regard to the background. For example, differences between API Ri Rm - API Ri X Cm represents the difference in intensity between the RNA spots and the background within the cytoplasmic region. Such differences may be useful parameters to monitor in a screening scenario, and, also are likely to be useful for optimization of the assay conditions and imaging parameters for particular samples types.

Nm is the nuclear mask and corresponds to the number of pixels that make up the nuclei. Cm is the cytoplasmic mask, which extends from the cell boundaries to the nucleus. Rm is the RNA mask and corresponds to the number of pixels found within RNA dots for the cell, as analyzed by CyteSeer-ViewRNA.

The first part of the data table portion of the C15_DataTable.csv file is shown in the figure above. Also shown is the analogous portion of the G15_DataTable.csv file. For this experiment, cells in the C15 well of the dish were not exposed to an activator of IL-8 expression. Thus, cells in C15 represent the negative control for the assay. Alternatively, cells in G15 were exposed to 1 ng/ml PMA, a phorbol ester that strongly activates IL-8 expression. For the first 10 cells analyzed for C15, no RNA spots were detected by CyteSeer ViewRNA . Thus, there are “0” values in Columns C, K, L, and M, which correspond to the data parameters area of the RNA mask (Area Rm), RNA spot count, and mean RNA spot area, and RNA spot diameter, respectively. Note, also that the first two cells of C15 were boundary cells (IsBoundaryNm = “True”), where as the rest of the cells were judged by CyteSeer ViewRNA as being entirely contained within the image (IsBoundaryNm = “False”). Data is reported on a total of 142 cells for well C15 in the C15_DataTable.csv file.

In contrast, all of the first 11 cells in the G15 data table featured RNA spots. Thus, there are positive data entries for every line in columns C, K, L, and M. For G15, cell number 8 (Excel line 44), for example, featured 2106 pixels in the RNA mask (column C), an RNA spot count of 148 (column K), a mean RNA spot area of 14.23 pixels (Column L), and an average RMS spot diameter of 4.2565 pixels. Note that data is reported on a total of 136 cells for well C15 in the C15_DataTable.csv file.

Key portions of the C15_DataTable_Stats.csv (found in the C15 directory) and the PMAvsIL8_DataTable_Stats.csv files (found under the parent directory for the experimental results are shown at right. The layout of the DataTable_Stats.csv files are related to, but somewhat different than the previously described DataTable.csv files. For example, values in column A are the StatsID numbers. There are 6 key statistics which are the Count (Row 39 in the C15_DataTableStats.csv file) which is the number of cells that were used in the calculations, the Mean, which is the average value obtained for all cells (the well population) that were analyzed in the well, Sigma, which is the standard deviation for the data parameter and for the well population, Median, which is the value of the data parameter for which 50% of the data values for the well exceeded (and 50% were below), the Min, which is the lowest value obtained, and the Max, which is the maximum value that was obtained. Column B displays the well designation for housekeeping purposes, and Column C displays the “Count”, “Mean”, “Sigma”, “Median”, and “Max” titles. Note that all of the data that is displayed refers to values that were derived on a “per cell” basis. For well C15, 142 cells were identified and the data that is summarized in the DataTable_Stats.csv files includes data derived from all of the cells (including the boundary cells), so the count is 142 for every statistic in the report. The Mean value for the RNA Spot Count for well C15 was 0.03521RNA Spots per cell (Excel address M40). Thus, very few RNA spots were identified by CyteSeer ViewRNA for well, C15, and a maximum of 2 spots per cell were found for the cell population.

Note that the PMAvsIL8_DataTable_Stats.csv file, features the identical display for well C15, along with data obtained from all wells in the experimental analysis. Thus, this file provides a convenient reference, displaying a summary of all the results for the experiment. The Experimentname_DataTable_Stats file will thus be one of the most frequently used data files generated by CyteSeer ViewRNA .

Results for the experiment in which the effect of PMA was tested on IL8 mRNA expression are shown in Figure 22. Results are graphed and tabulated for 3 key data parameters that describe mRNA expression. Area Rm, the average area, per cell, of the RNA mask as identified by CyteSeer ViewRNA was < 1 for well C15, but > 1100 for well G15. Thus, addition of 1 ng/ml PMA elicited a 3000-fold increase in this parameter. For the RNA spot count, essentially no spots were found for the control well (the average number of spots was approx. 0.04/cell), whereas 14.3 spots/cell were found for cells exposed to 0.1 ng/ml PMA (well E15), and 84.1 spots/cell were found for 1 ng/ml PMA (well G15). Also, note that the TII Ri Rm data parameter, which is the total intensity of the spots/cell, went up by 8000-fold. Since the Panomics Quantigene assay results in a single RNA spot per mRNA, the RNA Spot Count data parameter from CyteSeer ViewRNA will undoubtedly be of high interest to most users. Users interested in screening large chemical or siRNA libraries vs. mRNA expression, utilizing automated methodology, will also likely find the Area Rm and TII Ri Rm data parameters of high interest, due to the very high dynamic range these parameters may provide for the assay.

Once satisfied with the segmentation and statistics at an assay development level the CyteSeer ViewRNA settings can be applied on larger data sets.