LightCycler® Nano System Performance Data

LightCycler^® Nano System Performance Data

The LightCycler^® Nano Instrument looks great on your bench - and produces beautiful data. Like all Roche real-time PCR systems, the LightCycler^® Nano System uses excellent thermal control for high reproducibility of results obtained with the 32 samples tested in the same run. High resolution for copy number determination and melting curve analysis is achieved with great speed.

Here is a list of examples to illustrate these performance features. Click the ones that are most relevant for your application, and enjoy the data.

Topic	Template	Detection Format	Reagent
Dynamic range of quantification (2-fold dilutions)	plasmid DNA	Universal ProbeLibrary probe	FastStart Essential DNA Probes Master
Dynamic range of quantification (10-fold dilutions)	plasmid DNA	Universal ProbeLibrary probe	FastStart Essential DNA Probes Master
Reproducibility of Cq values	human genomic DNA	hydrolysis probe	FastStart Essential DNA Probes Master
Endpoint Genotyping	DNA	hydrolysis probe	FastStart Essential DNA Probes Master
High Resolution Melting	DNA	ResoLight dye	LightCycler^® 480 High Resolution Melting Master
Absolute Quantification	DNA	SYBR Green I	FastStart Essential DNA Green Master
Homogeneity of Tm values	human genomic DNA	SYBR Green I	FastStart Essential DNA Green Master

Dynamic range of quantification (2-fold dilutions)

Purpose of the experiment	Dynamic resolution of gene quantification using Universal ProbeLibrary probes.
Procedure	Real-Time PCR using 2-fold dilutions of plasmid DNA (starting from one million copies) as template, and a Universal ProbeLibrary probe for product detection.
Observation	The LightCycler^®Nano Instrument reliably discriminates template dilutions, even differences by a factor of 2, producing reproducible replicate Cq values.
Result	Serial amplification plots of 2-fold dilutions show the high resolution detection and differentiation.

Dynamic range of quantification (10-fold dilutions)

Purpose of the experiment	Dynamic resolution of gene quantification using Universal ProbeLibrary probes.
Procedure	Real-Time PCR using tenfold dilutions of plasmid DNA (1E0 to 1E9 copies) as template, and a UPL probe for product detection.
Observation	All dilutions are positive over the range of dilutions down to single copy detection. The LightCycler^® Nano Instrument reliably detects over 10 tenfold dilutions of template. Highly reproducible Cq values are produced . UPL probe results are, as expected, even more precise than those obtained in SYBR Green I assays.
Result	Serial amplification plots of Cq values from 10-fold dilutions indicate high PCR efficiency.

Cq homogeneity using hydrolysis probe

Purpose of the experiment	Homogeneity of results, independent of block position.
Procedure	Real-Time PCR using 1E4 template copies in each position. Beta-globin amplification from human genomic DNA using hydrolysis probe detection.
Observation	Excellent intra-run Cq homogeneity.
Result	Very low variation of Cq values (SD=0.02).

Fast, accurate endpoint genotyping

Purpose of the experiment	Show LightCycler^® Nano System capability for SNP endpoint genotyping.
Procedure	Genotyping analysis on MDR1 gene fragment, present in 3x 10 replicates. FastStart Essential DNA Probes Master kit was used for amplification and detection with hydrolysis probes.
Observation	The LightCycler^® Nano System differentiated wild type, heterozygote and mutant genotypes in an unbiased way.
Result	Scatter plot discriminates genotypes cleanly (blue=wildtype, green=mutant, red=heterozygote).

Fast, accurate detection of Class I SNPs using HRM

Purpose of the experiment	Show LightCycler^® Nano System doing high resolution melting analysis of Class I SNPs
Procedure	HRM analysis on a 149 bp fragment of the Adr-1 gene with an A to G variation. LightCycler^® 480 High Resolution Melting Master, including ResoLight Dye, was used for amplification and detection.
Observation	Excellent temperature accuracy of the LightCycler^® Nano System differentiates wild type, heterozygote and mutant genotypes in an unbiased way.
Result	Normalized melting curves (top), and difference plot (below), discriminate genotypes (blue=wildtype, green=mutant, red=heterozygote).

Accurate absolute quantification

Purpose of the experiment	Show LightCycler^® Nano System quantifying two different targets in one run using internal standard curves.
Procedure	Absolute quantification of two targets (CYP2C9 and CYP219) using internal standard curves. FastStart Essential DNA Green Master used for amplification and detection.
Observation	Both targets quantified with high accuracy in a single run.
Result	Amplification (A) and standard (B) curves for CYP2C9 (blue) and CYP2C19 (green), and unknowns (red) show accurate, reproducible quantification.

Tm homogeneity using SYBR Green I Detection

Purpose of the experiment	Verify homogeneity of Tm results, independent of block position.
Procedure	Real-Time PCR using 1E4 template copies in each position. Beta-globin amplification from human genomic DNA using SYBR Green I detection.
Observation	Excellent intra-run homogeneity.
Result	Very low variation of Tm values

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