The siRNA validation page gives an overview of the siRNA downregulation assay performed for the antibody. For each HPA antibody, the siRNA downregulation assay is performed in duplicates with two different siRNA:s. Only successful assays are included in the Atlas.
siRNA Validation score and downregulation details
In the table at the top the siRNA validation score for the assay is shown together with the results from the statistical analysis of the analyzed images. The Relative Fluorescence Intensity (RFI) denotes the percentage of remaining staining intensity and is calculated by comparing population median staining intensities between siRNA down regulation and scrambled (negative control) samples. Also, the cell segmentation compartment in which the changes in intensity have been measured is noted here. Depending on the original staining of the HPA antibody, a segmentation region covering either the nucleus, cytoplasm or the whole cell is used in the statistical analysis.
The boxplot on the top left is magnified when clicked, and graphically illustrates the changes in fluorescent light intensity between siRNA and scrambled samples.
siRNA downregulation assay images
siRNA transfected U-2 OS cells are immunofluorescently stained with the corresponding antibody. Images from the scrambled siRNA (negative control) well are displayed to the left and the images from one or two siRNA-assays are displayed to the right. Two 10x-images are included for each sample as well as where available one or two 40x images.
Use the antibody and intensity channel toggle (see below) to further illustrate the intensity downregulation when comparing images from scrambled and targeting siRNA samples in the images.
General information on images and viewing functionality
Three different organelle probes are displayed as different channels in the multicolor images - nuclear stain in blue, microtubules in red and ER in yellow. The antibody staining is shown in green. By using the "toggle channels"-buttons, the different channels can be turned on and off. The intensity toggle shows the pixel intensity range in 16 different colors, for the selected channel, antibody, nucleus, microtubules or ER.
To change which images to compare, drag and drop a miniature image from the bottom on one of the top three positions. Three images can be compared at a time.
All images are clickable for an enlarged view. The selected image will appear in large size and miniature images with all other staining results for this gene will be listed at the top left of the image. The selected miniature image is highlighted in orange.
