Effects of Plate Position, Plate Type and Sealing Systems on Real-Time PCR Results

ABSTRACT

Real-time PCR has become an important tool for quantitative nucleic acids analysis. By now, a wide rang of real-time PCR instruments, PCR plastic consumables and sealing systems are available. In this study both transparent and white plates were taken to test possible position and plate effects on quantitative PCR results on different real-time platforms: ep realplex (Eppendorf, Hamburg, Germany) and the iQ5 (Bio-Rad, Palo Alto, USA). Heat and adhesive sealing was used to compare sealing systems. Heterogeneity in Ct values was calculated to show possible effects of plate type and sealing system. Heterogeneity in amplification efficiency should show possible positional effects of 96-well plates.

White plates showed higher amplification efficiency because of higher fluorescence reflection. This had no significant effect on the PCR efficiency but on the sensitivity of the quantification assay. Constant positional effects concerning wells, columns or rows could not be detected.

Keywords:

• real-time qRT-PCR
• position effects
• plate type
• sealing system