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® Gene Quantification on the LightCycler® 480System Read in this article: General Concept LightCycler® 480 Gene Quantification Methods Key benefits of the LightCycler® 480 System for Gene Expression Analysis Absolute Quantification Relative Quantification/Gene Expression Analysis General Concept Currently, analysis of gene expression is a critically important area of scientific research. Manygenomic labs have found that quantitative real-time PCR is the gold standard for fast, sensitivedetermination of gene expression levels. However, not all quantitative real-time PCR techniquesare appropriate for gene expression analysis. There are many types and subtypes of quantitative real-time PCR methods, each of which ischaracterized by its requirements, its complexity, and its reliability. However, it is possible to groupall these methods under two main analysis techniques - absolute and relative quantification. Thetechnique you choose depends on the complexity of your analysis and the desired format of thefinal result:

Absolute quantification allows you to quantify a single target sequence and express the finalresult as an absolute value (e.g., viral load - copies/ml). Such analyses routinely occur inresearch areas like virology and microbiology.Relative quantification compares the levels of two different target sequences in a singlesample (e.g., target gene of interest (GOI) and another gene) and expresses the final result asa ratio of these targets. For comparison purposes, the second gene is a reference gene [aconstitutively expressed gene (housekeeping gene)] that is found in constant copy numbersunder all tested conditions. This reference gene, which is also known as endogenous control,provides a basis for normalizing sample-to-sample differences. Such analyses are useful, forinstance, in oncology research.

Back to Top LightCycler® 480 Gene Quantification Methods

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The LightCycler® 480 Software 1.5 offers several methods for precise relative quantification, eachof which handles the problem of PCR efficiency differently: CT-Method The CT-Method can provide fast quantitative data if the efficiency of the PCR assays for bothtarget and reference genes are optimal and/or identical. In cases where this prerequisite is notmet, the Roche -Method can provide more accurate data. The -Method The - (Efficiency)-Method can produce more accurate relative quantification data because it cancompensate for differences in target and reference gene amplification efficiency either within anexperiment or between experiments. It also enables normalization for run-to-run differences caused e.g., by variations in reagentchemistry. Basic Approach - Easy, convenient, fast, this method works with pre-defined settings, requiringno user input. It is an automated analysis, with one click to results. Advanced Approach - For highest flexibility and most demanding analyses, this approachprovides an analysis with extended options according to the user's needs. Advanced featuresinclude the use of multiple housekeeping genes for pairing.

one or several targets and/or reference genes can be usedaveraging of multiple housekeeping genes is possible (“all-to-mean” rule)editing of target-specific efficiency valuesflexible rules for pairing of target and reference genesanalysis of target and references present on same or different platesanalysis of full plates or plate subsetsFit Points Method or Second Derivative Maximum Method for Cp calling

Back to Top Key benefits of the LightCycler® 480 System for GeneExpression Analysis

Speed up PCR analysis with advanced, user-friendly, fast-tracking software tools.Easily customize data analysis with basic and advanced quantification methods.Facilitate multiple quantification analyses for a single PCR result.Produce high value quantitative real-time PCR data using proven LightCycler® algorithms.Achieve ultimate data accuracy with the -Method.

Figure 1: Result of an advanced relative quantification analysis using LightCycler® 480Software 1.5. (a) Upper part: results in table view, including sample information on chosen references, pairingand Cp values (b) Lower part: Bar-chart display (including errors) of target/reference ratios, with normalizedvalues in red. Back to Top Absolute Quantification Dual-color assay with hydrolysis probes and internal control

Figure 2: Absolute quantification assay with the LightCycler® 480 Instrument. Serially diluted standards (1:10 dilutions, three replicates each) were assayed along with unknownsamples. In addition, an internal PCR control (IC) was added to each sample to preventmisinterpretation of negative PCR results. The specific sequences were amplified with theLightCycler® 480 Probes Master and detected with hydrolysis probes (target: FAM-labeled, IC:VIC-labeled). Back to Top Relative Quantification/Gene Expression Analysis Relative quantification with the LightCycler® 480 Real-Time PCR System

Figure 3: Two different relative quantification analyses of the same run with the LightCycler® 480 System. RNA was reversely transcribed using the Transcriptor Reverse Transcriptase.Target cDNA sequences were amplified with the LightCycler® 480 Probes Master and detectedwith Universal ProbeLibrary Probes. This figure shows a typical target and reference run, withunknowns (in red or blue) and calibrator samples (in green), which are analyzed via either (a) theCT-Method or (b) the -Method. Whereas the data in (a) are based on an assumption that theefficiency () = 2, the -Method data in (b) are based on the true efficiency of each reaction, which isderived from serial dilutions of the target (light red, = 2.021) and reference genes (light blue, =1.966). The final results from (b) are automatically calculated from the Cp values of the target(unknowns and calibrator) and the reference (unknowns and calibrator); these results are depictedin (c).

LightCycler® 480 Reagents for Relative Quantification Click here for more information on Universal ProbeLibrary Probes or Sets. Click here for RealTime ready pre-tested assays and customized panels. > Reagents for 2-Step RT-PCR: Transcriptor First Strand cDNA Synthesis Kit LightCycler® 480 SYBR Green I Master LightCycler® 480 Probe Master for Hydrolysis probes, UPL probes, and Hybridization probes Find Reagents for 1-Step RT-PCR. Back to Top