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LightCycler® 480 System Performance Data

 

Read in this article:



Sensitivity

Linear range of the LightCycler® 480 Instrument

Figure 1: Serial 1:10 dilutions (nine replicates each) of a plasmid DNA sequence were amplified with the LightCycler® 480 Probes Master and detected with a UPL (Universal ProbeLibrary) probe. The PCR result shows a log-linear relationship over a broad dynamic range (10 log-intervals) and highly reproducible Cp values for replicates of each dilution.

 

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Two-fold resolution of the LightCycler® 480 Instrument

Figure 2: Serial 1:2 dilutions (seven replicates each) of a viral target sequence were assayed with the LightCycler® 480 SYBR Green I Master. A special pipetting scheme was used to distribute the samples across the entire plate. Reproducibility is shown by the uniformity of Cp values within replicate groups and low coefficients of variation (CV < 0.2 %).

 

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Two-step RT-PCR assay with SYBR Green I detection

Figure 3: Starting from a 100 ng/µl RNA stock solution, dilutions were made down to 10 pg/µl. 50 µl of each dilution were used for cDNA synthesis with Transcriptor First Strand cDNA Synthesis Kit in a reaction volume of 100 µl. 2 µl of the cDNA reaction were used in 20 µl PCR reactions to amplify the human HPRT target gene. The dilution series, including negative controls, was run in 16 replicates (two per row). Samples in the upper left and lower right parts of the plate were used to establish a standard curve, allowing to demonstrate that the obtained Cp values were highly reproducible across the plate.

 

Total RNA per reaction 100 ng 10 ng 1 ng 100 pg 10 pg
mean 20.57 24.11 27.91 31.69 35.49
SD 0.083 0.163 0.14 0.186 0.337

max

20.71 24.45 28.17 32.07 35.96

min

20.42 23.83 27.69 31.44 34.9

delta Cp

0.29 0.62 0.48 0.63 1.06

 

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Reproducibility

Low intra-assay variation of melting curve analysis across a 384-well plate

Figure 4: Three different samples of total human genomic DNA, encoding different variants of the MDR1 gene (two homozygous, one heterozygous), were added to a 384-well plate in a checkerboard pattern (96 replicates for each genotype). The target gene was then amplified and the product characterized by melting temperature analysis using the same primers and SimpleProbe probe for all variants. As demonstrated by the very low standard deviations (see table), the obtained melting temperature (Tm) values were highly reproducible.

 

 

Tm(1) °C

Tm(2) °C

average

47.97

58.31

delta

0.48

0.63

SD

0.1439

0.1597

 

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Reproducibility of absolute quantification with HybProbe probes

Figure 5: The Parvo B19 gene was amplified (four different concentrations of plasmid DNA, applied to a 384-well plate in a checkerboard pattern) and detected with a HybProbe probe pair. Samples in three corners of the plate (all except upper right) were used to establish a standard curve and compared to all other wells across the plate. As demonstrated by the very low standard deviations (see table), the obtained Cp values were highly reproducible.

 

 

Target copies per reaction

 

1.00E +05

1.00E +04

1.00E +03

1.00E +02

mean

20.45

24.19

27.67

31.04

SD

0.0993

0.1418

0.0920

0.2186

min Cp

20.53

23.9

27.45

30.58

max Cp

21.14

24.82

27.89

31.58

delta Cp

0.61

0.92

0.44

1.00

 

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Reagents

Stability of the LightCycler® 480 SYBR Green I Master at room temperature

Figure 6: Serial 1:10 dilutions (10,000 –10 copies/reaction, three replicates) of a human DNA target sequence were assayed either immediately after PCR set-up or after 24 hours standing in a loading robot at room temperature. The amplification curves and the corresponding Cps of the samples that were assayed immediately (blue curves) and those that were assayed later (red curves) demonstrate that the performance of the hot-start enzyme master mix was not affected by prolonged standing.

 

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Universal ProbeLibrary assay compared to commercial prevalidated assay

Figure 7: Universal ProbeLibrary assay compared to commercial prevalidated assay. Target: human GAPDH. Template: commercially available reference cDNA (stock concentration: equivalent of 50 ng/µl total RNA). Tested concentrations: 10-fold dilution series in triplicates; conc 1: 25 ng/20 µl, conc. 2: 2,5 ng/20 µl, conc. 3: 250 pg/20 µl, conc 4: 25 pg/20 µl, NTC: negative control. PCR kit: LightCycler® 480 Probes Master.

 

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Multicolor capabilities

Dual-color hydrolyis probe assays without color compensation

Figure 8: The indicated target genes (upper figure: PBGD and CYP 2C9, lower figure: HSV and G6PDH) were amplified from plasmid DNA (PBGD, CYP 2C9: 104 copies/well; HSV, G6PDH: 105 copies/well) in adjacent wells on the same plate and detected using hydrolysis probes labeled with the indicated dyes. One target and color were used per well, with each curve representing one well. The analysis of each well in all relevant channels showed absence of cross-talk for the investigated dual-color combinations (e.g., A, left panel:, pink curve: wells with CYP 2C9 and a RED 610-labeled probe are not excited in the FAM channel and do not give signal).

 

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