Multiplex PCR of 19 targets (99–955 bp) was performed using standard conditions for the QIAGEN Multiplex PCR Plus Kit, without further optimization (QIAGEN) or using a variety of concentrations of a hot-start DNA polymerase from Supplier AII. [A] Analysis using an agarose gel. [B] Analysis using the QIAxcel Advanced System. The QIAGEN Multiplex PCR Plus Kit resulted in specific amplification of all targets, without the need for optimization. Despite lengthy optimization using different enzyme concentrations, multiplex PCR using the kit from Supper AII resulted in absence of expected PCR products, even when using higher concentrations. M: GelPilot 100 bp Plus Ladder.
The QIAGEN Multiplex PCR Plus Kit was used to amplify 16 targets (99–955 bp) from various amounts of human genomic DNA, ranging from 1 ng to 1 µg. Successful multiplex PCR results were achieved in each case. M: GelPilot 100 bp Plus Ladder.
HeLa cDNA was either pre-amplified using the QIAGEN Multiplex Plus Master Mix according to the Supplementary Protocol (0.08 ng starting cDNA per qPCR), or non-amplified (1 ng cDNA per PCR). The QuantiFast SYBR® Green Kit was used to analyze the expression levels of 80 different genes that are involved in apoptosis pathways. A high correlation of CT values from unamplified samples versus pre-amplified samples was observed (R2=0.982), indicating even and non-biased amplification of all 80 genes in the pre-amplification step.