Pre-amplification in the context of high-throughput qPCR gene expression experiment

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.

The sceptical optimist: challenges and perspectives for the application of environmental DNA in marine fisheries

“…A number of different chips are available for this instrument (i.e. FLEXsix, 48.48 Dynamic Array and 96.96 Dynamic Array), which allow 12, 48 and 96 different assays and samples to be cross‐analysed simultaneously, respectively, enabling up to 9.216 qPCR reactions in a single run (Korenková et al., ; Spurgeon, Jones, & Ramakrishnan, ; Svec et al., ). Illustrated in another way, the 96.96 Dynamic Array plate, can potentially screen up to 96 different species in 96 samples simultaneously (excluding various non‐template controls and standards).…”

“…Illustrated in another way, the 96.96 Dynamic Array plate, can potentially screen up to 96 different species in 96 samples simultaneously (excluding various non‐template controls and standards). However, the platform uses small reaction volumes (<10 nl) requiring high concentrations of eDNA and the time required to optimize numerous assays to work under identical reaction conditions remain a major challenge for utilizing these platforms for eDNA analysis (Korenková et al., ; Spurgeon et al., ). It should also be noted that the assay design process is time‐consuming and can be complex due to the potential for cross‐reactivity to DNA from non‐target species and presently few species‐specific qPCR assays are designed for marine species.…”

“…In addition, bulk samples of eDNA from multiple individuals do not allow more advanced fisheries genetic analyses, such as population assignment tests (Nielsen, 2016), as these methods are individual-based, that is rely on the combined information from multiple genetic markers genotyped in the same individual. However, the platform uses small reaction volumes (<10 nl) requiring high concentrations of eDNA and the time required to optimize numerous assays to work under identical reaction conditions remain a major challenge for utilizing these platforms for eDNA analysis (Korenková et al, 2015;Spurgeon et al, 2015). It should also be noted that the assay design process is time-consuming and can be complex due to the potential for cross-reactivity to DNA from non-target species and presently few species-specific qPCR assays are designed for marine species.…”

Amy2Bcopy number variation reveals starch diet adaptations in ancient European dogs

“…Such pre-amplification procedures have been shown to improve the sensitivity of quantitative PCR (qPCR) analysis on modern [34,35] and aDNA [36]. We followed previous recommendations to perform robust and highly accurate targeted pre-amplification in combination with qPCR [34–36].…”

Highly Sensitive Detection of Low-Abundance White Spot Syndrome Virus by a Pre-Amplification PCR Method

“…However, the performance of DNA amplification is largely dependent on the conditions of the extracted template DNA [17]. When only trace amounts of target DNA are present in large amounts of background DNA, the probability of obtaining false-positive or false-negative results by DNA amplification may increase owing to nonspecific amplification or lower sensitivity, and therefore pre-amplification is frequently performed to enrich the target DNA before the final detection [18]. At the early stage of WSSV infection, only a few virus particles exist in numerous tissue cells; as a result, the concentration of WSSV DNA is extremely low in the extracted sample DNA, and hence challenges the accuracy of detection.…”