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Gene Quantification on the LightCycler® 480 System

 

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. Many genomic labs have found that quantitative real-time PCR is the gold standard for fast, sensitive determination of gene expression levels. However, not all quantitative real-time PCR techniques are appropriate for gene expression analysis.

There are many types and subtypes of quantitative real-time PCR methods, each of which is characterized by its requirements, its complexity, and its reliability. However, it is possible to group all these methods under two main analysis techniques - absolute and relative quantification. The technique you choose depends on the complexity of your analysis and the desired format of the final result:

  • Absolute quantification allows you to quantify a single target sequence and express the final result as an absolute value (e.g., viral load - copies/ml). Such analyses routinely occur in research areas like virology and microbiology.
  • Relative quantification compares the levels of two different target sequences in a single sample (e.g., target gene of interest (GOI) and another gene) and expresses the final result as a ratio of these targets. For comparison purposes, the second gene is a reference gene [a constitutively expressed gene (housekeeping gene)] that is found in constant copy numbers under 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, for instance, in oncology research.

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LightCycler® 480 Gene Quantification Methods

 

The LightCycler® 480 Software 1.5 offers several methods for precise relative quantification, each of which handles the problem of PCR efficiency differently:

 

ΔΔCT-Method

The ΔΔCT-Method can provide fast quantitative data if the efficiency of the PCR assays for both target and reference genes are optimal and/or identical. In cases where this prerequisite is not met, the Roche Ε-Method can provide more accurate data.

 

The Ε-Method

The Ε- (Efficiency)-Method can produce more accurate relative quantification data because it can compensate for differences in target and reference gene amplification efficiency either within an experiment or between experiments.
It also enables normalization for run-to-run differences caused e.g., by variations in reagent chemistry.

 

Basic Approach - Easy, convenient, fast, this method works with pre-defined settings, requiring no user input. It is an automated analysis, with one click to results.

Advanced Approach - For highest flexibility and most demanding analyses, this approach provides an analysis with extended options according to the user's needs. Advanced features include the use of multiple housekeeping genes for pairing.

  • one or several targets and/or reference genes can be used
  • averaging of multiple housekeeping genes is possible (“all-to-mean” rule)
  • editing of target-specific efficiency values
  • flexible rules for pairing of target and reference genes
  • analysis of target and references present on same or different plates
  • analysis of full plates or plate subsets
  • Fit Points Method or Second Derivative Maximum Method for Cp calling

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Key benefits of the LightCycler® 480 System for Gene Expression 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 Ε-Method.

Figure 1: Result of an advanced relative quantification analysis using LightCycler® 480 Software 1.5.
(a) Upper part: results in table view, including sample information on chosen references, pairing and Cp values
(b) Lower part: Bar-chart display (including errors) of target/reference ratios, with normalized values in red.

 

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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 unknown samples. In addition, an internal PCR control (IC) was added to each sample to prevent misinterpretation of negative PCR results. The specific sequences were amplified with the LightCycler® 480 Probes Master and detected with hydrolysis probes (target: FAM-labeled, IC: VIC-labeled).

 

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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 detected with Universal ProbeLibrary Probes. This figure shows a typical target and reference run, with unknowns (in red or blue) and calibrator samples (in green), which are analyzed via either (a) the ΔΔCT-Method or (b) the Ε-Method. Whereas the data in (a) are based on an assumption that the efficiency (Ε) = 2, the Ε-Method data in (b) are based on the true efficiency of each reaction, which is derived from serial dilutions of the target (light red, Ε = 2.021) and reference genes (light blue, Ε = 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 depicted in (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.

 

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