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Letters to the Editor |
Division of Human Genetics, University of Bern, Bern, Switzerland
aAddress correspondence to this author at: Division of Human Genetics, University Children’s Hospital, Inselspital, Freiburgstr., CH-3010 Bern, Switzerland. Fax 41-31-632-94-84; e-mail sabina.gallati{at}insel.ch.
To the Editor:
Carousel position-dependent effects on capillary-based LightCycler instruments have been described by Wilhelm et al. (1) and Zuna et al.(2). Both studies found such positional effects to depend on small temperature differences in the thermal chamber of the LightCycler that lead to variable DNA melting efficiencies when amplifying genomic DNA targets with high melting temperatures. These temperature inhomogeneities lead to carousel position-dependent differences in primer binding efficiency and thus to different cycles over threshold (Ct). Both authors postulated that such positional effects would disappear with the instrument’s software version 3.5 that allows constant rotation of the carousel, leveling off possible temperature inhomogeneities in the thermal chamber.
However, even when the use of software version 3.5 provided constant carousel rotation, we observed positional effects when amplifying a region of the SNRPN gene in an assay containing 26 reactions (performed on a LightCycler 1.2). Similar effects were observed with the ATP7B gene and the LightCycler Control Kit (Roche), and the effect was even stronger on a LightCycler 1.1 instrument. Position effects were also observed when only 10 reactions were performed. Of special concern is that the maximum Ct difference between different positions (
Ctmax) in the SNRPN assay was 
0.52 on the LightCycler 1.2 (CV of n replicates in a run 0.68%, n = 26) and 0.94 on the LightCycler 1.1 (CV 1.28%, n = 13) (Fig. 1
); such differences could indicate a duplication or a deletion in a diagnostic setup. Even more striking is that such position-dependent effects are usually not detected, as the sinusoidal shape of the effect causes only minor differences in Ct between neighboring samples.
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To investigate whether the effect we observed also depends on the melting temperature of the genomic target, we performed reactions using SNRPN PCR products as template (1), which resulted in the disappearance of the positional effect. Target melting temperature dependence of the observed effect was further supported by experiments demonstrating that a genomic region with lower melting temperature [CFTR exon 16(3)] was successfully amplified without any positional effect.
We hypothesized that the incomplete filling of the carousel in our assay with 26 reactions might cause airflow disturbances in the thermal chamber that prevent a homogeneous temperature distribution. We thus occupied the remaining positions of the carousel with liquid-filled capillaries, and the effect either completely disappeared (LightCycler 1.2; Ctmax 
0.16, CV 0.17%) or became weaker (LightCycler 1.1; Ctmax 0.71, CV 1.09%) (Fig. 1
), indicating that the filling status of the carousel plays a crucial role for the accuracy of the LightCycler instrument.
Further efforts were undertaken to improve performance of the LightCycler 1.1 instrument. Considering that our standard real-time PCR protocol has a denaturation step of 0 s at 95 °C during cycling (4), we supposed that increasing the denaturation time to 5 s would allow completion of 1 carousel rotation and thus adjust positional effects caused by temperature inhomogeneities in the thermal chamber. Indeed this measure resulted in a Ctmax of 0.11 and a CV of 0.16% (Fig. 1B
); however, this precision was achieved only when the carousel was completely filled; increasing the denaturation time to 5 s without filling the carousel resulted in a Ctmax of 0.39. Similarly, increasing the denaturation temperature (98 °C for 0 seconds) as well as denaturation for 5 s at 95 °C during the first 5 cycles only(1) did not generate the precision obtained with 5 s denaturation at 95 °C during all cycles. Finally, we found that the imprecision also varied slightly among carousels.
Based on our findings, we strongly recommend complete filling of the LightCycler carousel with capillaries, independent of the number of reactions performed, and setting the denaturation time to 5 s. Neglecting these conditions will generate false results with serious consequences for diagnostic investigations such as deletion and duplication screenings and detection of variations in copy number. Considering that there are only minor physical differences between the various LightCycler versions, we think that it is rather a coincidence that the observed effect was stronger on the LightCycler 1.1, and we advise to check any real-time PCR assay for eventual position dependent effects—not only on capillary-based cyclers, but also on plate-based platforms (5). Based on the fact that Pierce et al.(5) observed significant variations in Ct when using cDNA as template, we cannot exclude the possibility that positional effects also occur in mRNA analyses.
Acknowledgments
Grant funding/support: This work was supported by the Swiss National Foundation, Grants No. 3200-066767.01 and 310000-112652.
Financial disclosures: None declared.
Acknowledgments: We thank Dr. Lorenz Roggo and Prof. Dr. Rolf Jaggi for technical support and advice.
References
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