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Abstract
BackgroundWith 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. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. 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).ResultsFor 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 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted.ConclusionsWe 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. 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0033-9) contains supplementary material, which is available to authorized users.
Highlights
With the introduction of the first high-throughput Quantitative polymerase chain reaction (qPCR) instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds
Evaluating variables in pre-amplification reaction using regular qPCR instrument The primary purpose of pre-amplification is to enhance amount of input material, which can be, in some instances, very low even for conventional qPCR: single cell analysis [16,21], microRNA analysis [22], analysis of formalin-fixed, paraffine embedded tissues [23] or to enhance initial amount of material to be sufficient for high-throughput instrument [1]
The amount of preamplified transcripts correlates with the initial cDNA target copy numbers as has been shown previously for both good quality samples [24] and bad quality samples, e.g. formalin-fixed paraffin-embedded samples [23]
Summary
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. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. 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). 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 6.7 nl [2].
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