Supplemental Reference Guide

Supplemental Reference Guide QuantiGene ViewRNA mirna ISH Cell Assay P/N 19167 Rev.A 120623

For research use only. Not for use in diagnostic procedures. Trademarks Affymetrix and, and QuantiGene are trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners. Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a nonexclusive, non-transferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix. You understand and agree that, except as expressly set forth in the Affymetrix terms and conditions, no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product. In particular, no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided, licensed, or specifically recommended by Affymetrix for such use. Citing QuantiGene ViewRNA in Publications When describing a procedure for publication using this product, please refer to it as the QuantiGene ViewRNA mirna assay. Disclaimer Affymetrix, Inc. reserves the right to change its products and services at any time to incorporate technological developments. This manual is subject to change without notice. Although this manual has been prepared with every precaution to ensure accuracy, Affymetrix, Inc. assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. Copyright 2012 Affymetrix Inc. All rights reserved.

Contents Chapter 1 Introduction................................................... 1 About This Guide.........................................................1 Adjusting Intensity Thresholds and Overlaying Images............................1 Sample Images to Aid in Optimization and Multiplexing..........................2 Related User Documents....................................................2 Contacting Technical Support..............................................2 Chapter 2 Adjusting Intensity Thresholds and Overlaying Images................ 3 Procedure Using MetaMorph Software.........................................3 Procedure Using Image J Analysis Software.....................................10 Chapter 3 Example Images............................................... 15

iv QuantiGene ViewRNA ISH Tissue Assay User Manual

1 Introduction About This Guide This guide provides the following supplemental information for the QuantiGene ViewRNA mirna ISH Cell Assay: Procedure for using MetaMorph software to display acquired images, subtract non-specific background and overlay signals from different channels to generate merged images Procedure for using Image J software to display acquired images, subtract non-specific background and overlay signals from different channels to generate merged images Sample images to aid in optimization and multiplexing Adjusting Intensity Thresholds and Overlaying Images Similar to most immunofluorescence assays, raw images acquired through fluorescence microscopy for the QuantiGene View mirna Cell Assay will contain both specific and non-specific signals, making it difficult to visualize, compare and interpret results between samples. Being able to determine the background level of the negative control sample and subtracting it from all other images would provide a more accurate comparison and interpretation of the results within an experiment. This type of background subtraction requires the ability to adjust the contrast of an image, or specifically the black level of an image, otherwise known as "thresholding". There are several available imaging software applications that enable one to display and adjust the intensity thresholds of the image as well as overlay signals from different channels to generate a colorfully merged image for the purpose of publication or presentation. Here, we provide step-by-step instructions using two different software applications: MetaMorph imaging software, available for purchase from Molecular Devices Image J imaging software, available for free from National Institutes of Health (NIH) In the enclosed procedures, proper visualization requires, first and foremost, two sets of images-one for the negative control and the other for the experimental sample. Each set of images consists of data for the DAPI (nuclei), Cy3/FastRed (mirna) and FITC (mrna) channels and these are used as examples to demonstrate how the background for each of these channels is determined for the negative control and subtracted from the experimental sample to obtain specific signals that can then be overlaid to give a merged image. Please note that any adjustments made to the image intensity thresholds will only affect how the images are displayed and do not at all alter the absolute signal intensity values of the raw images, and thus do not impact the quantification of signal intensity should one choose to quantify the results. Image Quality Digital images to be adjusted can be taken with a variety of equipment. However, for the best result, capture and save images as Tagged Image File Format (TIFF). This format does not eliminate pixel information when compressed and hence retains the best resolution. Image Threshold Adjustment Workflow For both software programs, the workflow for adjusting thresholds and overlaying images is as follows: 1. Open (+) probe and (-) probe images for one channel. 2. Adjust upper threshold on (+) probe image to desired brightness. 3. Set upper threshold on (-) probe image to be the same. 4. Determine background and set lower thresholds for both images. 5. Repeat steps 1-4 for other channels. 6. Assign colors for each channel.

2 QuantiGene ViewRNA ISH Tissue Assay Supplemental Reference Guide 7. Overlay images from different channels. Sample Images to Aid in Optimization and Multiplexing Optimal pretreatment conditions for your cell type and proper assignment of mrna targets to the available channels when multiplexing ensure proper signal detection of your targets with maximal specific signal-to-background ratios when performing the QuantiGene ViewRNA mirna ISH Cell Assay. In the QuantiGene ViewRNA mirna ISH Cell Assay User Manual, we provide recommendations to empirically determine the proper pretreatment conditions for your specific cell type, criteria for selecting the optimized conditions, general trends to note in any pretreatment assay optimization study and guidelines for assignment of mrna targets to available channel when multiplexing. Here, in this supplemental guide, we provide sample images as visual guides to demonstrate both optimal and suboptimal pretreatment conditions, with the aim of assisting you in optimizing and troubleshooting your samples. Additionally, sample images of multiplexing mirna and mrna targets with different expression levels will also be shown to demonstrate proper assignment of mrna targets to the FITC and Cy7 channel. Related User Documents For QuantiGene ViewRNA mirna ISH Cell Assay procedures, see one of the following user manuals. These are available at www.affymetrix.com/panomics. QuantiGene ViewRNA mirna ISH Cell Assay User Manual for glass coverslips in 24-well plate QuantiGene ViewRNA mirna ISH Cell Assay User Manual for 96-well optical-bottom imaging plate Contacting Technical Support For technical support, contact the appropriate resource provided below based on your geographical location. For an updated list of FAQs and product support literature, visit our website at www.affymetrix.com/panomics. Table 1.1 Technical Support Contacts Location North America Europe Asia Contact Information 1.877.726.6642 option 1, then option 3; pqbhelp@affymetrix.com +44 1628-552550; techsupport_europe@affymetrix.com +81 3 6430 430; techsupport_asia@affymetrix.com

2 Adjusting Intensity Thresholds and Overlaying Images Procedure Using MetaMorph Software To adjust the threshold intensities using MetaMorph analysis software: Step Action 1 A. Open the MetaMorph image analysis software. B. Select File > Open and open the TIFF images for (+) probe and (-) probe for one channel such as Fast Red/ Cy3 or FITC. Note that the image histogram is displayed on the side of the image.

4 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide To adjust the threshold intensities using MetaMorph analysis software: Step Action 2 Using the upper slider, adjust the upper threshold of the histogram on the (+) probe image until spots are visible. Lowering the upper threshold will enhance the signal, where as increasing the upper threshold will diminish the signal. 3 Once the upper threshold for the (+) probe image has been adjusted to the desired brightness, apply the same upper threshold setting to the (-) probe image.

Chapter 2 Adjusting Intensity Thresholds and Overlaying Images 5 To adjust the threshold intensities using MetaMorph analysis software: Step Action 4 A. Determine the signal intensity of the background by pointing the cursor arrow to an area devoid of cells and nuclei. This value is indicated on the bottom of the screen. B. Adjust the lower threshold of the (-) probe and (+) probe image by dragging the lower sliders to a value (red arrows) that would eliminate background signals. Usually, this value is approximately 2-3X the signal intensity determined for the background. The same lower threshold setting should be used for both images. C. Save the images. 5 Repeat Step 1 to Step 4 for images taken in the other channels.

6 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide To adjust the threshold intensities using MetaMorph analysis software: Step Action 6 For the DAPI channel, open the raw (TIFF) DAPI images for the (-) probe and (+) probe samples. 7 A. Adjust the upper threshold for each image by dragging the upper sliders so that the nuclei are visually comparable between the two images and are not too bright or too dim. Because the DAPI staining can be variable between samples and serves only as a counterstain to identify placement of the cells, the upper threshold settings do not need to be the same between the (-) and (+) probe images. B. Dragging the lower sliders, set the lower thresholds to approximately 1X the background intensity. 8 Save and close the image files.

Chapter 2 Adjusting Intensity Thresholds and Overlaying Images 7 To adjust the threshold intensities using MetaMorph analysis software: Step Action 9 Open the adjusted image files for the channels you want to overlay. In this case, we are overlaying the Fast Red and DAPI channels for the (+) probe sample. 10 From the Display menu, select Overlay Images. The Overlay Image dialog box opens.

8 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide To adjust the threshold intensities using MetaMorph analysis software: Step Action 11 Enter the # Images to be overlaid. This value = N + 1, where N = the number of images (in this case 2). Therefore, the number to enter is 3, that is, 2 +1. NOTE: MetaMorph software assumes there is always a brightfield image, so 1 must be added to the overlaid images, even when you have no brightfield image. The Overlay Images dialog box now reflects three channels instead of five. 12 Assign hues to each image: A. Under Source, select one of image files. Since there are only two fluorescent image files and no brightfield image, the first source option will not be used. B. From the Hue slider bar, select a hue to represent the channel in this image file. In this example, we are choosing blue to represent DAPI and red to represent Fast Red. C. Repeat A and B for each image file. D. Adjust the balance by varying the number in the Bal column. This adjusts the intensity of the color. Use the overlay preview window (which appears when the color is assigned) as a guide for setting the balance.

Chapter 2 Adjusting Intensity Thresholds and Overlaying Images 9 To adjust the threshold intensities using MetaMorph analysis software: Step Action 13 Once the settings have been selected, click Apply to apply these settings to the images. It is important to apply the same balance settings to other images of the same channel and experiment in order to obtain a fair comparison of the signal, since a higher balance setting means the hue will be more vibrant and a lower setting means the hue will be more dim and muted (see below). Higher balance setting Lower balance setting 14 Save the overlaid image as a TIFF to preserve the highest resolution.

10 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide Procedure Using Image J Analysis Software To adjust the threshold intensities using Image J analysis software: Step Action 1 A. Open the Image J analysis software. B. Select File > Open and open the TIFF images for (+) probe and (-) probe for the channels you want to analyze, in this case DAPI and FITC. 2 From the Image menu, select Adjust > Brightness/Contrast. The B&C dialog box opens. 3 Working with the (-) probe and (+) probe images for the DAPI channel first, adjust the upper threshold for each image by dragging the slider on the Maximum scale in the B&C dialog box (see red circle below) either to the right (dimmer) or the left (brighter) so that the nuclei in both images are visually comparable and distinct. It is not necessary to have the upper threshold setting for the DAPI images to be the same.

Chapter 2 Adjusting Intensity Thresholds and Overlaying Images 11 To adjust the threshold intensities using Image J analysis software: Step Action 4 When you have adjusted the intensity between the (-) and (+) probe for DAPI, check the intensity of the background for each image: A. Place the arrow cursor to an area of the image devoid of cells or nuclei. The number is displayed as the value, as shown below, in this case 617. B. In the B&C dialog box, drag the slider on the Minimum scale to a setting that eliminates the background signal, approximately equal to the value of the background, 617. C. Repeat this step for both (+) and (-) probe images. Leave the images open.

12 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide To adjust the threshold intensities using Image J analysis software: Step Action 5 Leaving the DAPI images open, adjust the lower threshold (background) of the images of other channel, in this case FITC. A. Place the arrow cursor to an area in one of the FITC images devoid of cells or nuclei. The number is displayed as the value, as shown below, in this case 796. B. In the B&C dialog box, drag the slider on the Minimum scale to a setting that eliminates the background signal, approximately a value of 2-3X the background with the cursor, approximately 1700 in this case. C. Apply the same lower threshold setting to the other FITC image.

Chapter 2 Adjusting Intensity Thresholds and Overlaying Images 13 To adjust the threshold intensities using Image J analysis software: Step Action 6 A. In the (+) probe FITC image, adjust the upper threshold to the desired brightness and intensity. Drag the slider in the Maximum scale so that the signal is the desired intensity. Dragging to the right will lessen the intensity, to the left increase the intensity. B. Adjust the maximum and minimum display ranges of the (-) probe FITC image to match the (+) probe FITC image: 1) Click Set. The Set Display Range dialog box opens. 2) Enter the Minimum displayed value and Maximum displayed value and click OK. 3) Leave images open for the next step.

14 QuantiGene ViewRNA mirna Cell Assay Supplemental Reference Guide To adjust the threshold intensities using Image J analysis software: Step Action 7 Merge the DAPI and FITC channels for the (+) probe samples: A. Ensure all adjusted image files are open. B. From the Image menu select Color > Merge Channels. C. Assign a color to each channel by selecting the image file from the list. For instance, select (+) Probe_FITC.tif for Green and (+) Probe_DAPI.tif for Blue. D. Select None for Gray and Red. E. Deselect Create Composite and select Keep Source Images. Then click OK. A merged image displaying both the blue DAPI nuclei and green FITC signals appears. 8 Repeat Step 7 to generate a merged image for (-) probe images. 9 Save merged images as TIF format to preserve the highest resolution.

3 Example Images Examples representing a variety of results are provided to demonstrate both optimal and suboptimal pretreatment conditions in order to aid you in optimizing and troubleshooting your samples. Image Examples Under Fixation or Over Digestion with Protease Significant cell loss Poor mirna target retention Weak signals inside cells Fewer number of dots mirna (Let-7a) mrna (PPIB) Optimal Pretreatment Conditions mirna (Let-7a) mrna (PPIB) Good cell retention Good target retention Optimal probe accessibility Strong signals inside cells

16 QuantiGene ViewRNA mirna ISH Cell Assay Supplemental Reference Guide Image Examples Over Fixation or Under Digestion with Protease Poor probe accessibility Weak signals inside cells Fewer number of dots mirna (Let-7a) mrna (PPIB) Multiplexing mirna and mrna mir-17 (TYPE 1)-Fast Red PPIB mrna (TYPE 4)-488 Merged

Chapter 3 Example Images 17 Image Examples mi-30d (TYPE 1)-Fast Red PPIB (TYPE 4)-488 Merged mir-93 (TYPE 1)-Fast Red HPRT (TYPE 4)-488 Merged mir-107 (TYPE 1)-Fast Red ActB (TYPE 4)-488 Merged

18 QuantiGene ViewRNA mirna ISH Cell Assay Supplemental Reference Guide Image Examples Let-7a (TYPE 1)-Fast Red GAPD (TYPE 10)-740 Merged Let-7a (TYPE 1)-Brightfield (-) Probe Control (Merged)