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First Steps in Relative Quantification Analysis of Multi-Plate Gene RealTime ready Application Note June 2011 First Steps in Relative Abstract/Introduction Quantification Analysis This technical application note describes the first steps in data analysis for relative quantification of gene expression of Multi-Plate Gene experiments performed using RealTime ready Custom Panels. The easy initial setup as well as the “Result Export” Expression Experiments procedure of the LightCycler® 480 Software is described. Further preparation of the data depends on the means of analysis. Three routes to analyze the data are described: 1. Spreadsheet calculation software 2. GenEx Software from MultiD Analysis Heiko Walch and Irene Labaere 3. qbasePLUS 2.0 from Biogazelle Roche Applied Science, Penzberg, Germany Whereas GenEx and qbasePLUS are software packages from third party vendors specialized in RT-qPCR data analysis (1, 2) the spreadsheet calculation approach can be seen as a generic procedure for performing subsequent analysis either in the spreadsheet software or in other sophisticated statistical tools such as R, SigmaPlot, etc. (3, 4) Note that this article does not focus on qPCR setup, experimental planning or correct selection of reference genes. For details on this important topics, see publications such as the MIQE guidelines (5), or other recently published scientific best practices for qPCR expression studies (6, 7, 8). For life science research only. Not for use in diagnostic procedures. RealTime ready Setting Up the Experiment and Selecting the Assays A thoroughly planned experiment is vital to good scientific The gene list was generated using the focus list of assays for data. The RealTime ready Configurator (9) offers various genes “Amplified/overexpressed genes in cancers”, based on a possibilities to facilitate the creation of a reasonable list of publication from Santarius et al. (10). Additionally, some target genes. For example, the focus lists available under the genes from the NF-kB pathway were added via the “Search “Search by Focus Panel” function comprise RealTime ready by Pathway” function in the RealTime ready Configurator. assay collections for different fields of interest, such as: • Genes involved in various signaling pathways RealTime ready Custom Panel 384 – 96 (e.g., NF-kB, Jun, MAPK, sonic hedgehog, Notch, wnt) • Genes involved in specific biological processes (e.g., angiogenesis, oncology, apoptosis, induction of pluripotency, stem cell differentiation) • Genes belonging to certain families or groupings (e.g., protein kinases, phosphatases, proteases, transcription factors) These lists can be seen as scientifically sound suggestions, and the RealTime ready Configurator also offers the freedom to add and remove assays to the desired layout as needed. To demonstrate the first analytical steps in a multi-plate analysis, we set up an artificial time series experiment with two biological samples (S01 and S02) and user selected Replicates: 4 Reference two time points (T01 and T02) after treatment measured user selected Assays: 93 Genes: in duplicates for the cDNA synthesis step and a single qPCR user selected measurement each. With a total of 93 genes of interest Figure 1: Sample layout for a 384-well plate with 4 replicates of and 3 reference genes, we chose a 384-well plate layout with 93 targets of interest and 3 reference genes each. 4 replicates on each plate (see Figure 1). Performing the RealTime ready Experiment on the LightCycler® 480 Instrument RealTime ready Custom Panels can be customized on the the page contains a .zip file that can be downloaded and is RealTime ready Configurator (9). The selected assays are associated with the configuration. The archive contains two pre-plated, and ready to use on LightCycler® 480 Multiwell text files: Plates. Single assays are also available in liquid format. The 1. The sample editor import file with layout information “My Orders” page of the Configurator shows the current (see Figure 2) status of all orders. After an order is produced and shipped 2. The configuration info file with additional assay information (see Figure 3) General : Pos General : Target Name Rel Quant : Combined Sample/Target Type A1 HS|PRKCI|105941 Target Unknown Figure 2: An excerpt of the sample editor import file. The three columns contain information for the well coordinates (General:Pos), the contained A2 HS|AKT3|105933 Target Unknown assays (General:Target Name), and the type of the assay (Rel Quant: … … … Combined Sample/Target Type). The assay information is represented as a concatenation of the organism, the gene symbol, and the assay ID, all N5 HS|NFKB2|100649 Target Unknown separated by vertical dashes (“|”). The different types of assays are either a gene of interest (Target Unknown), a reference gene (Ref Unknown), or N6 HS|ALAS1|102108 Ref Unknown an assay from the control concept (Unassigned Unknown) not used for this demonstration. The file can be imported directly into the LightCycler® N7 HS|TRAF2|102966 Target Unknown 480 sample editor software or opened and modified in any text editor (for example, to change the reference genes or gene annotations) and then … … … reimported afterwards. 2 RealTime ready: First Steps in Relative Quantification Analysis of Multi-Plate Gene Expression Experiments Figure 3: Configuration info file containing assay annotation and Combined with the detailed assay information and annotations sequence information. The example configuration info file shown above available at the RealTime ready Configurator, all information required contains additional information regarding the plate layout, gene information, by the MIQE guidelines for scientific publications is provided (5). and assay details including the primer sequences. The contents of the archive greatly reduce the time needed The information on how the replicates on the plate are used to annotate and set up the experiments in the is plate-specific and also needs to be edited in the sample LightCycler® 480 Software. After creating a new experiment editor. It is essential to use consistent naming for samples, in the LightCycler® 480 Software (11), the provided file replicates, and any additional information that will be needed can be imported into the sample editor to assign all well and later to combine the data. assay information for this configuration (see Figure 4). Figure 4: Editing target and sample information using the sample editor import file in the sample editor of the LightCycler® 480 Software. Preparing and Exporting the Result Data from the LightCycler® 480 Software 1.5 The LightCycler® 480 Software offers several analysis The Cq values are generated using the “Abs. Quant Analysis” modules. For this example we want to evaluate the relative and the “automated second derivative” method (11). Finally expression levels of mRNAs in different biological samples. to do a meaningful comparison of different samples and To do this we compare the normalized Cq values which genes the Cq values need to be annotated with sample and can be used as a relative measurement for the abundance of gene information accordingly as demonstrated below. cDNA covered by the particular RealTime ready assay. RealTime ready: First Steps in Relative Quantification Analysis of Multi-Plate Gene Expression Experiments 3 Exporting the Result Data from the LightCycler® 480 Software In order to combine the results of multiple plates with tested with a standard cDNA sample, therefore missing values varying numbers of replicates, use absolute quantification can be interpreted as no expression of the relevant target in conjunction with the second derivative method for or, to be more precise, an expression beyond the level of Cq-calling. The actual relative quantification is done detection for that particular assay. For data analysis purposes, outside the LightCycler® Software, following standard those missing values can be dealt with in various ways, some RT-qPCR analysis procedures (11). Looking at the analysis of which will be explained briefly in the analysis sections. results, some well positions might be highlighted in green, If, however, there are any clear patterns visible, for example, indicating that no Cq value could be calculated. Such one single plate replicate or one column/row shows no calls “failures” may occur for many reasons and can be expected you should check the starting materials and the pipetting if large amounts of different assays are used with schemas for possible technical errors. uncharacterized samples. RealTime ready assays are function Figure 5: Export of the result Cq values in the LightCycler® 480 Software by right-clicking in the result table and saving the file to an appropriate location. After the “Abs. Quant. Analysis”, the result data needs to be exported by right-clicking in the result table in the analysis section of the LightCycler® 480 Software for every individual run (see Figure 5). For the sake of data quality, consider how and where to organize the data. In this context, having a dedicated folder structure, with for example, file names and/ or short experimental descriptions along with the result Figure 6: File and folder structure for Experiment. and IXO files names for a particular round of experiments, 1. Short experiment description; is a good start (see Figure 6). For this workflow example, 2. Sample editor import file; the experiment consisted of 3 LightCycler® 480 Instrument 3. Zip file provided by Roche containing the sample editor import file and runs saved as “result_CONFIG_ID_plateXYZ.txt”. assay annotation/information; 4 & 5. Exported result files for the individual plates. After exporting the results, the run and data gathering step is finished and the data needs to be cleaned and combined in order to be analyzed. 4 RealTime ready: First Steps in Relative Quantification Analysis of Multi-Plate Gene Expression Experiments General Considerations for Relative Quantification Expression Data Multi-plate multi-parameter analysis can be conducted in several ways; this note covers only the first steps of cleaning and combining the results. These steps are usually constant and independent of the level of sophistication followed in the statistical analysis afterwards. For a “fold change analysis” these steps are: 1.
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