Multiple Plate Gene Expression Analysis with the LightCycler® 480 Real-Time PCR System
Featured Project: Expression Analysis of Genes Involved in Neurodegenerative Disorders
Molecular Pathology Research and Development Laboratory, Department of Pathology, University of Tübingen, Germany
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Real-time PCR has become a standard tool for analyzing mRNA expression levels, e.g., for validation of data obtained from whole-genome approaches (e.g., microarrays). For data interpretation, however, real-time PCR instrument software mainly focuses on raw data and does not support analysis of multiple runs or plates. Software tools that improve and accelerate the exploitation and statistical analysis of extended studies are therefore needed.
We studied the expression of 16 transcripts that might be predictive for the progression stage of spinocerebellar ataxia 1 and 3 (SCA1 + 3), two autosomal-dominant neurodegenerative diseases. These transcripts had previously been selected based on microarray experiments because of aberrant expression patterns in comparison to reference genes. The LightCycler® 480 Multiple Plate Analysis Software was tested as a novel tool for the compiled analysis of multiple LightCycler® 480 Instrument runs performed during this study.
Materials and Methods
Universal ProbeLibrary-based real-time PCR assays were designed and carried out on the LightCycler® 480 System for all 16 targets and three reference genes. 27 human research samples reflecting various physiological conditions were analyzed for all targets using the LightCycler® 480 Relative Quantification algorithm. The individual result files were imported into the LightCycler® 480 Multiple Plate Analysis Software and combined to form a new study object. So-called properties (condition and stage) and corresponding values were defined (condition: SCA1 or SCA2; stage: mild or intermediate or severe), values assigned to each sample, and samples assembled in groups. The provided workflow option “RelQuant Summary” was chosen, and normalized ratios were selected to be displayed. A single Excel file, holding all the information associated with the data, was generated automatically by the software.
The workflow is summarized schematically in Figure 1.
Figure 1: Schematic of LightCycler® 480 Multiple Plate Analysis Software workflow for relative quantification analysis.
Results and Discussion
The Summary table in the Excel document (provided automatically by the software) combines the normalized ratios with standard deviations for all assays and all samples, together with their defined properties and assigned values, in a single sheet.The expression ratio distribution of all experiments carried out for each designed assay (=target gene) were obtained as box-and-whisker plots (see Figure 2).
Figure 2: Box plots of normalized ratios for "mild" and "severe" stage patients for 8 target assays analysed. The horizontal bars indicate the median, the box spans the inter quartile range, and the "whiskers" delineate the minimum and maximum of all data.
The question whether or not the expression of a transcript is different between the various tested conditions was addressed in the Criteria Comparison worksheet. For each assay and each value of any selected property, the average ratio plus standard deviation was obtained in tabular form and in a bar plot (see Figure 3).
Figure 3: Average ratios and standard deviations for all values within the selected property "stage". Additional filtering for the value "SCA1" of property "condition" was applied.
Compiled analysis of extended real-time PCR studies has often been hampered by the need to carry out multiple, error-prone, and time-consuming export-import or copy-paste operations into third-party applications. The LightCycler® 480 Multiple Plate Analysis Software now offers an efficient and easy-to-use tool, circumventing the risk of data loss or assignment errors and giving easy access to meaningful study results.