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The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time Taylor et al., 2019


As I am new to qPCR, I found this review article extremely helpful.


qPCR is a technique that provides precise and quantitative data on nucleic acids. The minimum information for publication of quantitative real-time PCR experiments (MIQE) provides guidelines for validation and data analysis procedures. Major sources of error are outlined in Taylor et al., 2019.


To validate a qPCR experiment and to minimize error and maximize data quality, you must first validate primers. To validate primers, an equalized pool of samples form each biological group is diluted 1:20 and initially tested suing a thermal gradient to determine the optimal annealing temperature, average level expression and unique product for each target from melt curve and gel analysis. The quantitative cycle (Cq) value from the optimal annealing temperature range can be used as a guide to establish the standard curve dilution factor for each target (i.e., if the Cq value for optimized temperature range is between 10 and 16, use a 1:8 serial dilution series of the pooled cDNA sample in water). An eight-point standard curve is tested for each primer pair using the same pooled sample and the appropriate dilution factor as determined from the thermal gradient data.





New paleo-temperature reconstructions indicate that the average temperature of the last few decades across temperate North America was the highest of any period in the past 1,500 years (NCA4, vol. 1, p.188). Depending on the region and scenario, temperatures are predicted to rise another 3.4–5.3°F (1.9–2.9°C) by mid-century (2036–2065) and 4.4–9.5°F (2.4–5.3°C) by late-century (2071–2100) (NCA4, Vol.I, p. 197, Fig. 3). In the contiguous United States, the Midwest is projected to have the highest increases and Southeast the lowest. Alaska’s surface temperatures are projected to continue to increase faster than the global mean (NCA4, Vol. 1, p. 305), which is particularly alarming considering the feedback potential of melting permafrost.


Plastic waste is a difficult global problem that is exacerbating, and being exacerbated by, climate change. New research has shown that as plastics break down, they also emit methane and ethylene, which are powerful greenhouse gases (Royer et al. 2018). In addition, as climate change impacts ocean circulation, this will impact the abundance and distribution of marine plastic pollution, of which the implications to coastal communities and ecosystems are still not well understood (Welden and Lusher 2017). Reduction of plastic pollution will benefit the climate and the health of people, fish, wildlife, and ecosystems.


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