miscript mirna PCR Array Data Analysis v1.1 revision date November 2014

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1 miscript mirna PCR Array Data Analysis v1.1 revision date November 2014 Overview The miscript mirna PCR Array Data Analysis Quick Reference Card contains instructions for analyzing the data returned from using the miscript mirna PCR Arrays and Assays. For more information on the bench procedure for these products, please refer to the appropriate user manual or handbook. For additional required assistance, please contact a local QIAGEN technical representative. Important Notes: Please complete all of work with the Data Analysis Web Portal in the same session. The data is not stored on a server, so all work is lost once the session (or the web browser) is closed. Be sure to export all processed data and results to an Excel file saved on a local computer. Please set the screen resolution to or greater, if possible. Step 1: Transferring data from instrument to data analysis portal The C T values must be exported from the qpcr instrument and formatted into an Excel spreadsheet. All of the plates/wells must be copied into a single worksheet. After the run is finished, calculate the C T value for each well using the real-time cycler software, as described in the following steps. 1. Define the Baseline The baseline is the noise level in early cycles, where there is no detectable increase in fluorescence due to PCR products. Ensure that baseline settings are the same across all PCR runs associated with the same experiment to allow comparison of results. a. For 96- and 384-well plates Use the Linear View of the amplification plot to determine the earliest visible amplification. Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification. Do not included cycles greater than 15 for the baseline. b. For the Rotor-Gene Q Use the Dynamic Tube setting along with the Slope Correct and/or Ignore First settings. For more information, refer to the Rotor-Gene Q User Manual. 2. Define the threshold The threshold should be set using a logarithmic amplification plot so that the log-linear range of the curve can be easily identified. Using the Log View of the amplification plot, place the threshold above the background signal but within the lower half of the log-linear range of the amplification plot. The threshold should never be set in the plateau phase. The absolute position of the threshold is less critical than its consistent position across PCR runs. Ensure that threshold settings are the same across all PCR runs in the same analysis to allow comparison of results. a. Various PCR instruments (such as Applied Biosystems models 7500 and ViiA 7, and Stratagene models Mx3005P and Mx3000P) may require adjustment of the default Manual C T threshold value of 0.2 to a lower value in order to analyze the data properly. Use a value of 0.02 as a starting point. b. For the Rotor-Gene Q, we recommend a C T threshold value of approximately 0.02 in order to analyze the data properly. 3. In the Fluidigm real-time PCR analysis software, ensure the Quality Threshold is set at Select Linear (Derivative) for Baseline Correction. Select User (Global) for the C T Threshold Method. We recommend setting the Threshold (EvaGreen) to as a starting point. Page 1 of 6

2 4. Export the resulting C T values for all wells to a blank MS Excel spreadsheet according to the manual supplied with the real-time PCR instrument for use with the miscript mirna PCR Array Data Analysis software. Step 2: Upload data 1. Open GeneGlobe Data Analysis Center in a web browser: view%20page/ 2. Choose the Experiment performed, the instrument used, and the catalog number(s) (if using arrays). If using the miscript mirna 96HC, miscript mirna mirbase Profiler, or miscript mirna mirbase Profiler 384HC plates, use the Please select dropdown menu to check all plates analyzed in the experiment. Click away from the dropdown menu and then click the Continue button. 3. Verify that the selections are accurately reflected. If not, click the Return to GeneGlobe Data Analysis Center link. 4. Format qpcr Data in Excel Spreadsheet using provided templates as models. a. All uploaded Excel files must place each Sample s C T values in a different column starting at Row 3, where each row represents an individual assay listed in the order in which they are located in the PCR plate. Label the Samples with a name in Row 1 and predefine the Groups in Row 2. The Group designations are optional, but if not defined, the cells in Row 2 should be blank. b. If starting with a 96-well plate, 100-well ring or 384-well plate which is in the 1x384 layout, the data needs to be arranged so that each raw C T value corresponds to the correct well location on the PCR Array. Place the first Sample into Column B. See the 96-Well Cataloged PCR Array or the 384-Well Cataloged PCR Array Excel file for an example template in which the data may be replaced. c. If starting with a 384-well plate which is in the 4x96 layout, then separate each Sample s data into different columns using the available 384-Well (4x96) Cataloged Array Patch Excel file for assistance. d. For the Fluidigm BioMark instrument and plates, separate each Sample s data into different columns using either the PCR_Array_96x96_Fluidigm_Patch or the PCR_Array_48x48_Fluidigm_Patch Excel file as appropriate for assistance. e. If using the miscript mirna 96HC, miscript mirna mirbase Profiler, or miscript mirna mirbase Profiler 384HC plates, or miscript mirna mirnome PCR Arrays, arrange the data as described above, and then append the data from each individual plate end-to-end / top-to-bottom in order of the individual plate catalog numbers. f. For Custom PCR Arrays, the data needs to be arranged so that each raw C T value corresponds to the order in which the assays are listed in the array layout. Place the first Sample into Column C. Place the list of assay catalog numbers from the Custom PCR Array signoff sheet in Column B. If the custom array content filled an entire 96-well or 384-well plate, see the respective 96-Well Custom PCR Array and the 384-Well Custom PCR Array Excel files for an example template in which the data may be replaced. If the custom content is replicated multiple times on a plate to characterize multiple Samples simultaneously, then use the Universal Custom PCR Array Patch Excel file to assist with separating each Sample s data into different columns. Page 2 of 6

3 g. For individual Assays, the data needs to be arranged so that the raw C T value corresponds to the order in which the assays are listed in the plate layout. Place the first Sample into Column C. Place the list of assay catalog numbers in Column B. See the Individual Assays Excel file for an example template in which the data may be replaced. 5. Click the Browse button and select the MS Excel file containing the PCR data form a local computer or server with a maximum number of 100 Samples. Click the "Upload" button. 6. The uploaded C T data is then displayed in the Uploaded Data window underneath the Analysis setup tab. Step 3: Analysis setup 1. Uploaded Data This page simply displays the data from the uploaded Excel file. 2. Sample Manager a. Assign individual Samples to relevant biological Groups, if not already done in uploaded Excel file. Assign each set of biological/technical replicate Samples to the same Groups using the dropdown menus next to each Sample name. b. Select housekeeping mirnas to be used for normalization calculations for Custom miscript mirna PCR Arrays ONLY. i. Scroll through the custom mirna list and select the desired reference mirna by clicking it. To select multiple options in Windows, hold down the control (ctrl) key; in Macs, hold down the command key. ii. Click the Add button. The reference mirna will appear in a new window to the iii. right. If needed, select reference mirna in the new right-hand list and click the Remove button to delist them. c. PreAmped Sample Dilution Fold If miscript PreAMP PCR Kit was used, select the recommended dilution used in the experiment from the dropdown menu. d. Cell-Free Sample Source i. Select the Yes radial button if such Samples were used. ii. Identify whether the internal control was used and whether to use it for normalization using the appropriate radial buttons. If Yes is selected for the latter option, a new Processed Data link and page appears under the Analysis setup tab displaying the intermediate calculation correcting all C T values by the internal control C T values. e. Lower Limit of Detection The values are automatically set based on the selected instrument. If preamplification has been performed, adjust the lower limit of detection as indicated. Otherwise, do not adjust unless recommended by Technical Support. f. Click the Update button after all desired changes have been made on this page. 3. Data QC a. This page displays the results of a quality control check on each Sample. b. If all controls on all Samples passed the QC criteria, the displayed table will simply indicate that fact. c. If not, a summary table of the Samples and the controls that failed will be displayed complete with raw C T values and the threshold values for passing QC. If any control returns as Inquiry, then check the Troubleshooting guide in the appropriate miscript mirna PCR Handbook or call Technical Support at Page 3 of 6

4 4. Select Normalization Method a. Choose your preferred normalization method from the dropdown menu using the provided descriptions to help guide the choice. b. Click the Perform Normalization button. 5. Data Overview If desired, review data for the Distribution of C T values and the Percent Distribution of C T Values as well as a bar chart of the latter in each defined Group. Also, review the raw C T values, Average C T values, and their standard deviation ( ST DEV ) across the Samples in each defined Group. Simply select the desired Group from the dropdown menu and click the Update button. Step 4: Analysis i. Fold Regulation and Fold Change a. Fold Change is calculated as 2^- C T where: i. C T is the difference between each assay s C T value and the normalization factor from the chosen method. ii. C T is the difference between the C T values of each defined Test Group and the Control Group. b. Fold Regulation is calculated as the negative inverse of any Fold Change value less than one. Fold Change values greater than one are also the Fold Regulation. c. On both pages, the p-values are calculated based on a Student s t-test of the replicate 2^(- C T ) values for each mirna in the Control Group and Test Groups. d. Also on both pages, Comments are also provided if any Fold Regulation or Fold Change value should be interpreted with caution. Click the Comments link for further explanations. ii. iii. Average C T This page displays the intermediate calculation of the average normalized C T values for every mirna across the Samples in the each Group based on the chosen normalization method. 2^- C T This page displays the intermediate calculation of the average normalized expression levels for every mirna across the Samples in the each Group. Step 5: Plots & charts 1. Scatter Plot and Volcano Plot The Scatter Plot graphs the log 10 of each mirna s normalized expression in one Group versus another Group with boundary lines defining a fold-change threshold. The Volcano Plot graphs the log 2 fold change in each mirna s expression between two Groups versus the -log 10 of the p-value for each fold change value. a. Define Groups to be compared using the Y Axis and X Axis dropdown menus in the Scatter Plot or the Test and Control dropdown menus in the Volcano Plot. b. Choose the desired fold-change Boundary (or threshold) for both plots and the p-value threshold for the Volcano Plot. c. If desired, select a different Color Scheme from the dropdown menu. d. Click check boxes to remove mirnas from or add mirnas to the plots. To highlight a mirna s symbol on the plot, mouse over the right-hand table entries. Page 4 of 6

5 e. After any new changes to the selections have been made, click the Update button to view the new plot. f. Click the down arrow icon in the upper right corner of the plot to choose a method to save the image of the plot. g. Click the Export Data button to download the results as an Excel file. 2. Clustergram The Clustergram performs an unsupervised hierarchical clustering of the data to group mirnas together with similar patterns of expression. The default settings are recommended for ease of interpretation. However, the follow options may be adjusted. a. Sample: Plot horizontal axis by "Array" (Sample) or by "Group". b. Dimension: Cluster in "1-D" by mirnas only or in "2-D" by mirnas and Samples. c. Join Type: Choose Minimum, "Average", or Maximum for shorter to longer dendograms, respectively. d. Color coded: Select whether the color coding range reflects data from "Genes" (mirnas), "Samples" or the "Entire Dataset". e. Item names visible: Select whether or not to display the mirna symbols and Array (Sample) or Group names. f. Click check boxes in the right-hand table to remove mirnas from or add mirnas to the plot. g. After any new selections have been made, click the Update button to view the new plot. 3. Heat Map NOTE: This feature is not available for Custom PCR Arrays and Primer Assays. The Heat Map displays color-coded fold change results in the layout of the array and a table listing the mirna ID and the Fold-Regulation results used in to plot the Heat Map, and any Comments (along with their explanations) from the Fold Regulation and Fold Change pages under the Analysis tab. a. Define Groups to be compared and whether to report the fold-change results as a log transformation or not. The default setting selecting the transformation is recommended. b. Click the "Update" button once changes have been made to view the new Heat Map. c. Click the Export Data button to download the results as an Excel file. 4. Multigroup Plot a. Click check boxes to add mirnas to or remove mirnas from the plot (maximum of 10). b. Choose the results to plot on the y-axis using the radial buttons, either "AVG delta(c T )", "2^-delta(C T )", or "Fold Change". c. Choose the Chart Type from the dropdown menu, either Line Chart or Column Chart. d. Use the checkbox to Show Uncertainty error bars or not. e. Click the Show Chart button. f. Click the down arrow icon in the upper right corner of the plot to choose a method to save the image of the plot. Step 6: Export data 1. Be sure that the datasets desired for export are selected with the checkboxes. 2. Click the Export button to save an Excel spreadsheet containing the datasets in separate worksheets. Page 5 of 6

6 Step 7: What s next 1. mirna Expression a. Select the Control Group and the Experimental Group to be compared using the dropdown menus. b. Select whether interested in mirna with Decreased Expression, Increased Expression, or Both. c. Enter the desired fold regulation and p-value cut-off thresholds into the appropriate dialog boxes. d. Click the Show Selected mirnas button. e. View the mirnas that met the selected criteria with their fold regulation values and p- values. f. Select the appropriate radial button to download the data and available assay catalog numbers in an Excel file format suitable for ordering Individual Assays, Custom PCR Array, or Mimics and Inhibitors. For Custom PCR Arrays, then select the number of unique assays to analyze in the same array from the options available in the dropdown menu. g. Click the Export Data to download the Excel file. 2. Gene Regulation a. Select the Control Group and the Experimental Group to be compared using the dropdown menus. b. Select whether interested in mirna with Decreased Expression, Increased Expression, or Both. c. Enter the desired fold regulation and p-value cut-off thresholds into the appropriate dialog boxes. d. Click the Show Selected mirnas button. e. View the mirnas that met the selected criteria with the fold regulation values, p-values, and individual assay catalog numbers with links to their product pages. f. Click the Search for Regulated Genes button. g. View the genes along with the selected mirna predicted to regulate them, data about the predictions, and the individual RT 2 qpcr Assays, and links to their product pages, to analyze the expression of the regulated genes. Genes predicted to be regulated by upregulated and down-regulated mirna are listed in separate tables. h. Click the Export List of Genes button to download an Excel file containing all of the data from both tables. miscript mirna PCR Arrays, miscript mirna PCR Assays, and miscript mirna Data Analysis are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor. Page 6 of 6