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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

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The LightCycler

480 Software 1.5 offers several methods for precise relative quantification, each
of 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 both
target and reference genes are optimal and/or identical. In cases where this prerequisite is not
met, the Roche -Method can provide more accurate data.


The -Method

The - (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 -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
CT-Method or (b) the -Method. Whereas the data in (a) are based on an assumption that the
efficiency () = 2, the -Method data in (b) are based on the true efficiency of each reaction, which is
derived 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 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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