All these systems are designed to significantly simplify experimental workflow, increase throughput and reduce costs, while providing excellent data quality. There are only a few high-throughput qPCR instruments on the market that can be compared with BioMark System: OpenArray using a chip with 3,072 reactions, each for 33-nanolitre reaction volumes (Life Technologies) and SmartChip with 5,184 reactions, each for 100-nanolitre reaction volumes (Wafergen). With this number of reactions in a single run and its versatility and the freedom of the custom designed assays, BioMark System outperforms other high-throughput qPCR systems. The BioMark System is able to process a high number of reactions (9,216) in a single run, each reaction taking place in volume of 6.7В nl. Microfluidic Dynamic Arrays provided by Fluidigm are able to combine either 48 samples with 48 assays or 96 samples with 96 assays in a combinatorial manner inside the integrated fluidic circuit (IFC). The first commercially available high-throughput qPCR instrument was the BioMarkв„ў System from Fluidigm that was launched in 2006. Several years ago, the boom in high-throughput instruments changed the way of studying gene expression and enabled researchers to perform large scale studies based on the most sensitive and specific quantitative PCR method. The popularity of real time PCR steadily increases as well as the number of platforms, detection chemistries and multiple choices of analytical methods.

We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step. We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success.

The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20В ng of total RNA with 15В cycles of pre-amplification, 20x and 40x diluted and 5В ng and 20В ng of total RNA with 18В cycles of pre-amplification, both 20x and 40x diluted. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. ResultsДЇor evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078В ng, 0.32В ng, 1.25В ng, 5В ng, and 20В ng of total RNA). Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds.

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